Abstract
Is it time to rethink the inoculum of animal models of implant-associated infections (IAI)? Traditionally, animal models of IAI are based on inoculation with metabolically active overnight cultures of planktonic bacteria or pre-grown surface-attached biofilms. However, such inoculums do not mimic the clinical initiation of IAI. Therefore, the present study aimed to develop a clinically relevant inoculum of low metabolic micro-aggregated bacteria. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth (TSB) for seven days to facilitate the formation of low metabolic micro-aggregates. Subsequently, the aggregated culture underwent filtration using cell strainers of different pore sizes to separate micro-aggregates. Light microscopy was used to evaluate the aggregate formation and size in the different fractions, while isothermal microcalorimetry was used to disclose a low metabolic activity. The micro-aggregate fraction obtained with filter size 5–15 μm (actual measured mean size 32 μm) was used as inoculum in a porcine implant-associated osteomyelitis (IAO) model and compared to a standard overnight planktonic inoculum and a sham inoculum of 0.9 % saline. The micro-aggregate and planktonic inoculums caused IAO with the re-isolation of S. aureus from soft tissues, bones, and implants. However, compared to their planktonic counterpart, neither of the micro-aggregate inoculated animals showed signs of osteomyelitis, i.e., sequester, osteolysis, and pus at gross inspection. Furthermore, inoculation with low metabolic micro-aggregates resulted in a strong healing response with pronounced osteoid formation, comparable to sham animals. In conclusion, the formation and separation of low metabolic bacterial micro-aggregates into various size fractions is possible, however, planktonic bacteria were still seen in all size fractions. Inoculation with micro-aggregates caused a less-aggressive osteomyelitis i.e. combination of infected tissue and strong healing response. Therefore, the use of low metabolic micro-aggregates could be a relevant inoculum for animal models of less-aggressive and thereby slower developing IAI and add in to our understanding of the host-implant-bacteria interactions in slow-onset low-grade infections.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 100200 |
| Tidsskrift | Biofilm |
| Vol/bind | 7 |
| Antal sider | 12 |
| ISSN | 2590-2075 |
| DOI | |
| Status | Udgivet - jun. 2024 |
Bibliografisk note
Funding Information:This study was financed by grant no. R345-2020-1674 from the Lundbeck Foundation and grant no. NNF19OC0056411 from the Novo Nordisk Foundation, Challenge Program.
Funding Information:
S. aureus strain S54F9 (spa-type t1333), originally isolated from a porcine lung abscess was used in the present study. This strain was chosen since it is known to cause infections in porcine models [ 23\u201325], including osteomyelitis in a highly used IAO porcine model [ 26\u201328]. The strain has previously been whole-genome sequenced [22] and is capable of biofilm formation [29]. Tryptone Soya Broth (TSB) has previously been reported to support S. aureus aggregation [30]. Biofilm micro-aggregates were produced by culturing S54F9 in 400 ml TSB (Oxoid CM0129B, Oxoid Ltd, Basingstoke, United Kingdom) in a shaking incubator at 37 \u00B0C and 150\u2013180 rpm. for seven days. Planktonic overnight cultures of S54F9 were grown in Lysogeny Broth (LB) (Difco no. 240230, Becton, Dickinson and Company, New Jersey, USA) and TSB in a shaking incubator at 37 \u00B0C and 150\u2013180 rpm. for 24 h. The planktonic bacteria cultured in LB has previously been used as planktonic inoculum in the IAO porcine model [ 26\u201328] and was therefore used as planktonic inoculum in the present study. The planktonic bacteria cultured in TSB were included for comparison of differences between the different growth media and culturing times.This study was financed by grant no. R345-2020-1674 from the Lundbeck Foundation and grant no. NNF19OC0056411 from the Novo Nordisk Foundation, Challenge Program.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Louise Kruse Jensen reports financial support was provided by Lundbeck Foundation. Thomas Bjarnsholt reports financial support was provided by Novo Nordisk Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Publisher Copyright:
© 2024 The Authors