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Research Article

Interkingdom Signaling Induces Streptococcus pneumoniae Biofilm Dispersion and Transition from Asymptomatic Colonization to Disease

Laura R. Marks, Bruce A. Davidson, Paul R. Knight, Anders P. Hakansson
Melinda M. Pettigrew, Invited Editor, Larry S. McDaniel, Editor
Laura R. Marks
Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, New York, USAa
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Bruce A. Davidson
Department of Anesthesiology, University at Buffalo, State University of New York, Buffalo, New York, USAb
Department of Medicine, Veterans Affair Medical Center, Buffalo, New York, USAc
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Paul R. Knight
Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, New York, USAa
Department of Anesthesiology, University at Buffalo, State University of New York, Buffalo, New York, USAb
Department of Medicine, Veterans Affair Medical Center, Buffalo, New York, USAc
The Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York, Buffalo, New York, USAd
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Anders P. Hakansson
Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, New York, USAa
The Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York, Buffalo, New York, USAd
New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, New York, USAe
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Melinda M. Pettigrew
Yale University
Roles: Invited Editor
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Larry S. McDaniel
University of Mississippi Medical Center
Roles: Editor
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DOI: 10.1128/mBio.00438-13
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  • FIG 1 
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    FIG 1 

    Establishment of a pneumococcal biofilm model on live epithelial cells. S. pneumoniae D39 or EF3030 bacteria were seeded on paraformaldehyde-fixed HRECs. At 48 h, biofilms were transplanted to live HRECs. (A and B) Biofilm biomass (A) and sensitivity to gentamicin (500 µg/ml) (B) were measured by viable counts over time. Green lines indicate bacterial biofilms on fixed epithelia, and blue lines represent the bacteria after transplantation to live epithelial cells. (C) Representative three-dimensional (3D) reconstruction of confocal image of a D39 biofilm at 48 h posttransplantation onto live HREC layers. Bacteria are stained red using Baclight Red, and epithelia were stained with 488-conjugated concanavalin A (green) and their nuclei with DAPI (blue). The numbers represent distances in micrometers. (D) Representative image from the midcell confocal section view of a D39 biofilm sample after 48 h on live HRECs showing no bacterial invasion. HRECs were stained with anti-secretory component (SC) antibody (red), nuclei were stained with DAPI (blue), and pneumococci were stained with anti-PspA antibody (green).

  • FIG 2 
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    FIG 2 

    Dispersion of biofilm bacteria by virus infection and host factors. (A) The ATP concentrations in the supernatant of HRECs infected with influenza A virus (IAV) or in the nasopharyngeal lavage fluid recovered from mice 24 h after IAV infection were determined. (B) The impact of IAV infection on the ratio of bacteria in the supernatant (dispersed) to epithelium-associated bacteria (biofilm) 48 h after biofilm transplant to live HRECs was measured. (C) The change in the optical density at 600 nm (ΔOD600) of biofilm supernatant was measured 2 h after application of the indicated stimuli (NE = norepinephrine). The change in optical density correlated well with the number of CFU obtained from bacterial growth of the same supernatants. For each analysis, three independent assays were performed in duplicate. Statistical analysis was performed using Student’s t test (* = P < 0.05, ** = P < 0.01, *** = P < 0.001).

  • FIG 3 
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    FIG 3 

    Characterization of pneumococcal populations for gene regulation, adherence, invasion, toxicity, and cytokine induction from exposed HRECs. Biofilm and dispersed and planktonic pneumococcal populations show distinct gene expression profiles of key virulence genes (P < 0.05 for all comparisons using 2-way analysis of variance [ANOVA]) (A) and differences in transparent- and opaque-phase states (*** = P < 0.001 using 1-way ANOVA). (C to F) The populations further differed in their ability to adhere to (C), invade (D), and kill (E) HRECs (** = P < 0.01 for all comparisons using 1-way ANOVA) and induce different levels of key cytokines involved in proinflammatory responses from the exposed HRECs (* = P < 0.05 using 2-way ANOVA) (F). Each experiment was repeated at least three times in duplicate. ns, nonsignificant.

  • FIG 4 
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    FIG 4 

    Colonization and dissemination of pneumococcal populations after intranasal inoculation, aspiration inoculation, or intraperitoneal inoculation. (A and B) Individual 6-week-old female BALB/cByJ mice were inoculated with biofilm (designated “B”) or dispersed (designated “D”) or broth-grown, planktonic (designated “P”) populations of EF3030 (A) and D39 (B) pneumococci intranasally without anesthesia to determine nasopharyngeal colonization and dissemination from the nasopharynx to the lungs and middle ears. Additionally, the bacterial burden in nasopharyngeal (NP) lavage fluid was determined. (C and D) Mice were furthermore inoculated with EF3030 (C) and D39 (D) pneumococci intranasally after anesthesia in an aspiration pneumonia model, and the bacterial burden in the lung tissue and dissemination to the bloodstream were determined by plate counts. (E and F) Finally, each bacterial population of EF3030 (E) and D39 (F) pneumococci was injected intraperitoneally to determine the respective levels of virulence upon reaching the vascular compartment. For both nonanesthetized and anesthetized intranasal challenges, nasal lavage fluids and all tissues were collected at 48 h postinfection. For intraperitoneal challenge, samples were collected at 24 h postinfection. Each dot in the graphs represents an individual mouse. An “X” represents a mouse that became moribund and required euthanasia before the end of the experiment. Each experiment included at least 6 mice. Statistical analysis was performed using one-way ANOVA (* = P < 0.05, ** = P < 0.01, and *** = P < 0.001, indicating significant differences between the populations).

  • FIG 5 
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    FIG 5 

    Histological representation of mouse tissues infected with various pneumococcal populations. Representative histological images of nasal epithelium, middle ear space, and lungs 7 days following nonanesthetized intranasal challenge (rows 1 to 3) with PBS (mock-infected control), biofilm, or dispersed or planktonic populations. Row 4 shows histological sections of the lungs 48 h following intratracheal aspiration of these same bacterial populations. Tissues were stained with hematoxylin and eosin and examined microscopically at magnifications of ×400 for row 1 and ×200 for rows 2 to 4. The sections are labeled with relevant structures (LI, leukocyte infiltrate; TM, tympanic membrane; SM, stapes muscle; ME, middle ear cavity; Spn, S. pneumoniae).

  • FIG 6 
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    FIG 6 

    Bacterial dissemination of mice stably colonized with pneumococci and challenged with influenza A virus. Bacterial burden in tissues and nasopharyngeal lavage fluid was measured for individual 6-week-old female BALB/cByJ mice colonized intranasally without anesthesia with EF3030 (A and C) or D39 (B and D) biofilm bacteria at 1 (A and B) or 5 (C and D) days postinfection with IAV. Each dot represents an individual mouse. Statistical analysis was performed using Student’s t test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

  • FIG 7 
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    FIG 7 

    Bacterial dissemination of mice stably colonized with pneumococci and challenged with dispersants. The bacterial burden in tissues and nasopharyngeal (NP) lavage fluid was measured for individual 6-week-old female BALB/cByJ mice colonized intranasally without anesthesia with biofilm bacteria from (A) EF3030 or (B) D39 pneumococci for 48 h and then challenged intranasally with 20 µl PBS, 10 mM ATP, 100 nM norepinephrine (NE), or 1 M glucose (Glu) or subjected to 4 h of febrile-range hyperthermia (FRH). Each experiment represents at least three individual experiments with duplicate samples. Statistical analysis was performed using Student’s t test, comparing treatment conditions to PBS control. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Supplemental Material

  • Figures
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  • Figure S1 

    Colonization and dissemination of bacteria passively released from pneumococcal biofilms. Data represent tissue tropism of bacteria passively released from EF3030 (A) and D39 (B) biofilms during normal growth after 48 h. Mice were challenged via nonanesthetized intranasal (Colonization) and intraperitoneal (Sepsis) inoculation. For intranasal challenges, nasal lavage fluids and the respective tissues were collected at 48 h postinfection. For intraperitoneal challenge, samples were collected at 24 h postinfection. Bacterial burden was determined by viable counts of the respective fluid or tissue homogenate samples. Statistical analysis of this data, using Student’s t tests, showed no significant differences between bacterial burdens of biofilm bacteria and those reported in the legend to Fig. 4. Download Figure S1, TIF file, 0.2 MB.

    Copyright © 2013 Marks et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Table S1 

    Oligonucleotides used in qRT-PCR analysis. Table S1, DOCX file, 0.1 MB.

    Copyright © 2013 Marks et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Additional Files

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  • Supplementary Data

    Supplementary Data

    Files in this Data Supplement:

    • Text so1, TIF - Text so1, TIF
    • Text so2, DOCX - Text so2, DOCX
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Interkingdom Signaling Induces Streptococcus pneumoniae Biofilm Dispersion and Transition from Asymptomatic Colonization to Disease
Laura R. Marks, Bruce A. Davidson, Paul R. Knight, Anders P. Hakansson
mBio Jul 2013, 4 (4) e00438-13; DOI: 10.1128/mBio.00438-13

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Interkingdom Signaling Induces Streptococcus pneumoniae Biofilm Dispersion and Transition from Asymptomatic Colonization to Disease
Laura R. Marks, Bruce A. Davidson, Paul R. Knight, Anders P. Hakansson
mBio Jul 2013, 4 (4) e00438-13; DOI: 10.1128/mBio.00438-13
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