In Vivo Expression of Streptococcus pyogenes Immunogenic Proteins during Tibial Foreign Body Infection

Abstract

dGroup 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.

Immunoproteomic Identification ofIn Vivo-Produced Propionibacterium acnes Proteins in a Rabbit Biofilm Infection Model

“…Most of the immunogenic proteins identified in this study (Table 2) are known to have a metabolic function in bacteria. This phenomenon of proteins with metabolic functions having antigenic potential has been observed in other immunoproteomic studies with P. acnes (9,38) and other Gram-positive pathogenic strains, such as S. aureus (27) and S. pyogenes (18). We focused our immunoproteomic analysis on cell wall-and membrane-associated proteins of P. acnes, since previous studies have shown that these proteins may be able to be used as novel diagnostic biomarkers or vaccine candidates (39).…”

“…We chose to develop a comparatively easy and representative model of musculoskeletal infection and used this model to examine the protein expression of P. acnes during a chronic infection. Our model was a rabbit osteomyelitis model established for S. aureus by Mader and Shirtliff (17) and was successfully adapted for A. baumannii (our unpublished data) and S. pyogenes (18) by using an intramedullary injection of P. acnes-infected dextran beads to provide an abiotic surface for colonization and biofilm growth.…”

“…We used a rabbit tibial implant infection model that was adapted from Shirtliff et al (16) and Mader and Shirtliff (17) for other bacterial species, including Streptococcus pyogenes (18), Staphylococcus epidermidis, and Acinetobacter baumannii (our unpublished data).…”

“…New Zealand White female rabbits (Charles Rivers Laboratory, Wilmington, MA), 8 weeks of age and weighing 1.5 to 2.0 kg, were anesthetized, and a bacterial infection was introduced according to a previously described method (18). In short, an 18-gauge needle was inserted into the intramedullary cavity of the left tibial metaphysis, and then 0.1 ml of a 5% (wt/vol) solution of morrhuate sodium (Eli Lilly, Indianapolis, IN), 0.1 ml of the P. acnes mixture (inoculum described above), and 0.2 ml of sterile saline were sequentially injected.…”

Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

“…1-3 RHD is a worldwide problem and it is more common in Asian developing countries due to poor health facilities. [4][5][6] The usual detection methods of RHD infection are culture test, 7 biochemical test 7,8 impedimetric immunosensor, 9 multiplex PCR and genetic markers, [10][11][12] illumigene kit assay 13 and fluorescent in situ hybridization (FISH). 14 All the above methods are time taking, less sensitive and specific, costly and non-confirmatory based on a single test.…”