Streptococcus pneumoniae forms surface-attached communities in the middle ear of experimentally infected chinchillas.
BACKGROUND
Streptococcus pneumoniae (pneumococcus) causes respiratory and systemic infections that are a major public health problem worldwide. It has been postulated that pneumococci persist in vivo in biofilm communities.


METHODS
In this study, we analyzed whether pneumococci form biofilms in vivo, and if so, whether biofilms correlated with bacterial persistence. Chinchillas were infected with S. pneumoniae TIGR4 and euthanized at varying times after infection, after which the superior ear bullae were excised and examined by culture and microscopy.


RESULTS
Dense material, resembling the biofilms of other otitis media pathogens, was visible in the middle ear as late as 12 days after infection. Scanning electron microscopy revealed bacteria within an electron-dense matrix, similar to pneumococcal biofilms formed in vitro. Viability staining revealed groups of viable diplococci, as well as viable and nonviable host cells, attached to a fibrous matrix that was positive when stained with propidium iodide. Cryosections of biofilms were treated with polyclonal antibodies against the pneumococcal surface components pneumococcal surface protein A family 2, pneumococcal surface protein C, choline-binding protein, and neuraminidase, coupled with appropriate secondary antibody conjugates. Immunofluorescent staining showed the presence of pneumococcal communities within the material recovered from the middle ear chamber.


CONCLUSIONS
On the basis of these data, we conclude that pneumococci form biofilms in vivo and that this process may be intertwined with the formation of neutrophil extracellular traps. These findings provide new insights into the potential causes of antibiotic treatment failure and bacterial persistence in chronic pneumococcal otitis media.
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA.
Sean D
SD
Reid
Wenzhou
W
Hong
Kristin E
KE
Dew
Dana R
DR
Winn
Bing
B
Pang
James
J
Watt
David T
DT
Glover
Susan K
SK
Hollingshead
W Edward
WE
Swords
D000818:Animals; D018441:Biofilms; D002470:Cell Survival; D002682:Chinchilla; D004195:Disease Models, Animal; D004432:Ear, Middle / Q000382:microbiology* / Q000473:pathology / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D011018:Pneumonia, Pneumococcal / Q000635:transmission*; D013296:Streptococcus pneumoniae / Q000254:growth & development* / Q000648:ultrastructure