A modified chronic infection model for testing treatment of Staphylococcus aureus biofilms on implants.

Nis Pedersen Jørgensen,Rikke Meyer,Frederik Dagnæs-Hansen,Kurt Fuursted,Eskild Petersen,Herminia De Lencastre

doi:10.1371/journal.pone.0103688

Nis Pedersen Jørgensen, Rikke Meyer + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0103688

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Abstract

Bacterial biofilms causing implant-associated osteomyelitis is a severe complication with limited antimicrobial therapy options. We designed an animal model, in which implant associated osteomyelitis arise from a Staphylococcus aureus biofilm on a tibia implant. Two bioluminescently engineered (luxA-E transformed), strains of S. aureus were utilized, Xen29 and Xen31. Biofilm formation was assessed with epifluorescence microscopy. Quantitative measurements were performed day 4, 6, 8, 11 and 15 post-surgery. Bacteria were extracted from the biofilm by sonication and the bacterial load quantified by culturing. Biofilm formation was evident from day 6 post-implantation. Mean bacterial load from implants was ∼1×104 CFU/implant, while mean bacterial load from infected tibias were 1×106 CFU/bone. Bioluminesence imaging revealed decreasing activity throughout the 15-day observation period, with signal intensity for both strains reaching that of the negative control by day 15 while there was no significant reduction in bacterial load. The model is suitable for testing antimicrobial treatment options for implant associated OM, as treatment efficacy on both biofilm and viable counts can be assessed.

Highlights

The incidence of implant-associated osteomyelitis (OM) is increasing, with approximately 100,000 cases of OM each year in the United States of America, and an infection rate of 5–15% in fracture-fixation devices and 0.3–1% in joint-prosthesis [1,2,3]
Staphylococci are the most common bacteria isolated in implantassociated OM, with Staphylococcus aureus accounting for 35% and coagulase-negative Staphylococci for 40% [1]
The homogenization did not affect bacterial viability, as median log Colony Forming Units (CFU)/ml did not differ between freezing compared to freezing + homogenization; 4.5 log CFU/ml, range 4.2–4.6 log CFU/ml vs 4.5 log CFU/ml, range 4.4–4.6 log CFU/ml and 3.3 log CFU/ml, range 3.2–3.3 log CFU/ml vs 3.2 log CFU/ml, range 3.2–3.3 log CFU/ml

Summary

Introduction

The incidence of implant-associated osteomyelitis (OM) is increasing, with approximately 100,000 cases of OM each year in the United States of America, and an infection rate of 5–15% in fracture-fixation devices and 0.3–1% in joint-prosthesis [1,2,3]. Infected implants colonized by biofilm-forming bacteria are very difficult to diagnose and treat effectively [4]. Biofilms are communities of sessile bacteria enclosed in an extracellular matrix. Compared to the planktonic phenotype, bacteria of the biofilm phenotype are resistant to the host immune response and to antimicrobial treatment [2,3]. The increased resistance is partly caused by a lower metabolic rate in biofilm bacteria, and the embedding of bacteria in an extracellular polymer matrix [4,5,6,7]. Besides increasing resistance against antibiotics, the matrix confers mechanic stability and ensures the close proximity between bacteria, allowing cell-tocell communication and exchange of genetic material, both of which is vital to the biofilm [10,11]

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