Mechanisms of Persistence, Reactivation and Eradication of Staphylococci in Orthopaedic Infections
| dc.contributor.advisor | Atkins, Gerald J | |
| dc.contributor.advisor | Richter, Katharina | |
| dc.contributor.author | Zelmer, Anja Ruth | |
| dc.contributor.school | School of Medicine : Orthopaedics and Trauma | en |
| dc.date.issued | 2024 | |
| dc.description.abstract | Incidence rates of osteomyelitis are rising, leading to substantial burdens for healthcare systems and individuals, a reduced quality of life, and elevated risk of morbidity and mortality. Chronic and recurrent infections have the highest risk of treatment failure and present therefore a major challenge. The predominant pathogen Staphylococcus aureus is highly adaptable, and can persist in niches such as biofilms, abscesses, the osteocyte lacuna-canaliculi network and intracellularly, creating barriers that impede the effectiveness of both immune responses and antibiotics. Furthermore, S. aureus can adapt its phenotype to reduce antibiotic susceptibility, for example as a viable but non-culturable (VBNC) phenotype. Osteocytes, the most abundant and long-lived bone cells, are potential long-term reservoirs for S. aureus. This led to the hypothesis that S. aureus can persist within osteocytes for extended periods by transitioning into a VBNC state, shielded from antibiotic treatments and immune defences. A systematic review of treatments targeting intracellular S. aureus in osteomyelitis revealed a significant knowledge gap, particularly concerning intra-osteocytic and chronic infections. Additionally, many models did not account for clinically achievable antibiotic dosages. To address this, I first investigated whether an existing osteocyte-like Saos-2 infection model could be augmented, by accelerating the osteocytic differentiation process. This was achieved by using 1% O2 instead of atmospheric O2 levels, leading to a similar osteocytic differentiation state after 14 days, compared to 28 days. With this differentiation model, I investigated acute and chronic intracellular infections of S. aureus in Saos-2 osteocyte-like cells, which were treated for 1 or 7 days at dosages that were achievable within bone tissue. Rifampicin, levofloxacin and linezolid reduced bacterial colony forming numbers in the acute model. However, no treatment influenced bacterial mRNA expression, nor could it prevent the regrowth of bacteria, after reaching a non-culturable state. In a review for an international consensus meeting, we defined criteria for a rigorous in vitro model for intracellular infections in osteomyelitis and a systematically reviewed existing in vitro models. To further investigate chronic intracellular infections of S. aureus in osteocytes, we investigated multiple bacterial, host-cell and environmental factors. During chronic intracellular infections of S. aureus, bacterial culturability decreased more compared to DNA quantification and RNA expression, indicating a transition to a VBNC state. This was confirmed by imaging at a non-culturable state. Furthermore, the influence of the host cell type (primary vs cell line), bacteria strain, ability to form biofilms and to survive at lysosomal pH levels, phenotypical appearance and oxygen levels on chronic osteocyte infections in vitro were investigated. We conclude that S. aureus can persist in vitro intracellularly in osteocytes, for at least 21 days, adapting to a VBNC state, but retaining the ability to regain culturability. Some antibiotic treatments can reduce culturable bacterial numbers, but these do not eradicate S. aureus. Therefore, the bacterial intra-osteocytic adaptation of S. aureus to a VBNC phenotype may explain the non-culturability of patient samples and the antibiotic treatment failures in chronic osteomyelitis, and presents an ongoing clinical challenge for the successful treatment of bone infections. | en |
| dc.description.dissertation | Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School : Orthopaedics and Trauma, 2024 | en |
| dc.identifier.uri | https://hdl.handle.net/2440/141584 | |
| dc.language.iso | en | en |
| dc.provenance | This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals | en |
| dc.subject | osteomyelitis | en |
| dc.subject | S.aureus | en |
| dc.subject | intracellular infection | en |
| dc.subject | VBNC | en |
| dc.title | Mechanisms of Persistence, Reactivation and Eradication of Staphylococci in Orthopaedic Infections | en |
| dc.type | Thesis | en |
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