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, 82 (9), 3891-9

In Vivo Expression of Streptococcus Pyogenes Immunogenic Proteins During Tibial Foreign Body Infection

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In Vivo Expression of Streptococcus Pyogenes Immunogenic Proteins During Tibial Foreign Body Infection

Jeffrey A Freiberg et al. Infect Immun.

Abstract

Group A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilm in vivo was visualized using peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressed in vitro under biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with the in vivo results, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.

Figures

FIG 1
FIG 1
Left (infected) and right (uninfected) tibia from rabbit.
FIG 2
FIG 2
Stained histologic sections from infected rabbit tibia. (A) H&E-stained dextran bead (asterisk) surrounded by inflammatory infiltrate. (B) Gram-stained S. pyogenes microcolonies (arrows) within damaged bone.
FIG 3
FIG 3
Histologic sections from infected rabbit tibia stained with PNA-FISH FITC-labeled Universal bacterial probe. (A) Dextran bead (asterisk) surrounded by S. pyogenes biofilm (arrows). (B and C) S. pyogenes microcolonies and chains of cocci (arrows) adherent to lacunae within bone.
FIG 4
FIG 4
Representative 2DGE and Western blots of group A streptococcus cell wall protein. (A) 2DGE—biofilm cell wall protein. (B) Western blot—biofilm cell wall protein. (C) 2DGE—planktonic cell wall protein. (D) Western blot—planktonic cell wall protein. Note that some spots listed in Table 2 are not present, as not all spots were visible in every sample at every time point.

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