Streptococcus pneumoniae in Biofilms Are Unable to Cause Invasive Disease Due to Altered Virulence Determinant Production

Abstract

It is unclear whether Streptococcus pneumoniae in biofilms are virulent and contribute to development of invasive pneumococcal disease (IPD). Using electron microscopy we confirmed the development of mature pneumococcal biofilms in a continuous-flow-through line model and determined that biofilm formation occurred in discrete stages with mature biofilms composed primarily of dead pneumococci. Challenge of mice with equal colony forming units of biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Biofilm pneumococci of numerous serotypes were hyper-adhesive and bound to A549 type II pneumocytes and Detroit 562 pharyngeal epithelial cells at levels 2 to 11-fold greater than planktonic counterparts. Using genomic microarrays we examined the pneumococcal transcriptome and determined that during biofilm formation S. pneumoniae down-regulated genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide (CPS) production, and virulence. We confirmed these changes by measuring CPS by ELISA and immunoblotting for the toxin pneumolysin and the bacterial adhesins phosphorylcholine (ChoP), choline-binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP). We conclude that biofilm pneumococci were avirulent due to reduced CPS and pneumolysin production along with increased ChoP, which is known to bind C-reactive protein and is opsonizing. Likewise, biofilm pneumococci were hyper-adhesive due to selection for the transparent phase variant, reduced CPS, and enhanced production of PsrP, CbpA, and ChoP. These studies suggest that biofilms do not directly contribute to development of IPD and may instead confer a quiescent mode of growth during colonization.

Adherence and Biofilm Production of Streptococcus pyogenes

“…Our assumption was approved in this study, especially for highly invasive isolates, which were low and unstable biofilm producers during tested periods of incubation. Similar results for invasive isolates were observed for S. pneumoniae [33,34]. According to these results we could assume that for invasive isolates biofilm production is not crucial virulence factor.…”

“…Several authors have noticed better biofilm production in non-invasive streptococcal strains compared to invasive strains [33,34], and also in erythromycin-sensitive isolates compared to erythromycin-resistant isolates of S. pyogenes [35]. These results strongly indicate that biofilm production is protective mechanism enabling bacterial survival of antibiotic treatment and immune system reaction.…”

Mutations in Pneumococcal cpsE Generated via In Vitro Serial Passaging Reveal a Potential Mechanism of Reduced Encapsulation Utilized by a Conjunctival Isolate

“…Pneumococcal strains with reduced levels of capsular polysaccharide were previously shown to more effectively form biofilm structures and adhere to host epithelial cells (10,15,69). In support of these findings, the AI45dup-containing strain exhibited an enhanced ability to form static biofilms in vitro and adhere to a human epithelial cell line.…”

Pneumococcal Interactions with Epithelial Cells Are Crucial for Optimal Biofilm Formation and Colonization In Vitro and In Vivo

“…Antimicrobial resistance is a general characteristic of biofilms (17,25,41,59). To further characterize the phenotype of S. pneumoniae during nasopharyngeal carriage in the mouse, we evaluated the susceptibility of colonizing pneumococci to both the bactericidal antibiotic gentamicin and the more commonly used antibiotic penicillin and compared that to their sensitivity in vitro.…”

“…Although it has been speculated that pneumococci colonizing the nasopharynx may form biofilms (50,52,59,67,73), thus far biofilm formation in vivo has been shown only during disease states and has been found to various degrees in association with adenoids and mucosal epithelia of children with recurrent or chronic middle ear infections and chronic rhinosinusitis (33,36,57,60,61). Bacterial aggregates have also been documented in the lungs of infected animals (60).…”