Streptococcus pneumoniae Biofilm Formation Is Strain Dependent, Multifactorial, and Associated with Reduced Invasiveness and Immunoreactivity during Colonization
Authors: Blanchette-Cain Krystle , Hinojosa Cecilia A. , Akula Suresh Babu Ramya , Lizcano Anel , Gonzalez-Juarbe Norberto , Munoz-Almagro Carmen , Sanchez Carlos J. , Bergman Molly A. , Orihuela Carlos J. 
Published: October 2013

Biofilms are thought to play an important role during colonization of the nasopharynx by
Streptococcus pneumoniae
, yet how they form
in vivo
and the determinants responsible remain unknown. Using scanning electron microscopy, we show that biofilm aggregates of increasing complexity form on murine nasal septa following intranasal inoculation. These biofilms were highly distinct from
in vitro
biofilms, as they were discontiguous and appeared to incorporate nonbacterial components such as intact host cells. Biofilms initially formed on the surface of ciliated epithelial cells and, as cells were sloughed off, were found on the basement membrane. The size and number of biofilm aggregates within nasal lavage fluid were digitally quantitated and revealed strain-specific capabilities that loosely correlated with the ability to form robust
in vitro
biofilms. We tested the ability of isogenic mutants deficient in CbpA, pneumolysin, hydrogen peroxide, LytA, LuxS, CiaR/H, and PsrP to form biofilms within the nasopharynx. This analysis revealed that CiaR/H was absolutely required for colonization, that PsrP and SpxB strongly impacted aggregate formation, and that other determinants affected aggregate morphology in a modest fashion. We determined that mice colonized with Δ
psrP
mutants had greater levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1β, and KC in nasal lavage fluid than did mice colonized with wild-type controls. This phenotype correlated with a diminished capacity of biofilm pneumococci to invade host cells
in vitro
despite enhanced attachment. Our results show that biofilms form during colonization and suggest that they may contribute to persistence through a hyperadhesive, noninvasive state that elicits a dampened cytokine response.

IMPORTANCE
This work demonstrates the first temporal characterization of
Streptococcus pneumoniae
biofilm formation
in vivo
. Our results show that the morphology of biofilms formed by both invasive and noninvasive clinical isolates
in vivo
is distinct from that of formed biofilms
in vitro
, yet propensity to form biofilms
in vivo
loosely correlates with the degree of
in vitro
biofilm formation on a microtiter plate. We show that host components, including intact host cells, influence the formation of
in vivo
structures. We also found that efficient biofilm formation
in vivo
requires multiple bacterial determinants. While some factors are essential for
in vivo
biofilm formation (CiaRH, PsrP, and SpxB), other factors are less critical (CbpA, LytA, LuxS, and pneumolysin). In comparison to their planktonic counterparts, biofilm pneumococci are hyperadhesive but less invasive and elicit a weaker proinflammatory cytokine response. These findings give insight into the requirements for and potential role of biofilms during prolonged asymptomatic colonization.