Characterization of clinical S. aureus isolates from implant-associated infections with regard to adhesion to and invasion into osteoblasts
Implant-associated infections are severe complications in orthopaedic surgery. Beneath S. epidermidis, S. aureus is the most prevalent pathogen responsible for orthopaedic infection. Treatments using antibiotics are often unsuccessful due to biofilm formation or the intracellular persistence of S. a...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2022
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| Zusammenfassung: | Implant-associated infections are severe complications in orthopaedic surgery. Beneath S. epidermidis, S. aureus is the most prevalent pathogen responsible for orthopaedic infection. Treatments using antibiotics are often unsuccessful due to biofilm formation or the intracellular persistence of S. aureus. In the literature, few reports have taken S. aureus as a facultative intracellular pathogen. This study focuses on adhesion, invasion and intracellular survival of clinical isolates of S. aureus on/in osteoblasts.
Isolates obtained from implant-associated infection were determined by API® Staph. test and 16S rRNA sequencing. Further characterization was carried out using clear zone formation, collagenase and oxidase test, growth analysis, antibiotic resistance and biofilm formation analysis. For bacteria-osteoblasts interaction studies, S. aureus ATCC 29213 and eight S. aureus isolates (Pat 2, 4, 6, 9, 28, 36, 42, 45), as well as confluent layers of osteoblast like cell lines SaOS2 and MG63 were used.
Standard infection model was established using S. aureus ATCC 29213 to infect SaOS2 and MG63. Based on this model, adhesion and invasion assay with S. aureus isolates were processed. MTT and respiration assay were performed to get the metabolic activity results of the infected osteoblasts. PCR and qPCR were applied to identify genes involved in adhesion and the corresponding expression level. Mass spectrometry was used for quantitative protein analysis. Induced and inhibited autophagy, Western blot were carried out to analyse the role of autophagy in bacteria-osteoblasts interactions.
Based on the characterization and sequence analysis from the perspective of homology, isolates Pat 2, 4, 6, 9, 28, 36, 42, 45 were classified as S. aureus. In the infection model, a significant stronger adherence of S. aureus ATCC 29213 to SaOS2, rather than MG63, was observed. Bacteria harvested in stationary growth phase showed a stronger adherence to osteoblasts as compared to bacteria harvested in the logarithmic growth phase. Three S. aureus isolates (Pat 36, 42, 45) were found to adhere to and invade in osteoblasts strongly. PCR showed that eno, fnbA, clfA, clfB, ebpS, psmA, psmB, eap, sdrD, fmtB and ebh genes were expressed in all S. aureus isolates. Furtherly, qPCR showed that clfA might be a candidate gene related to bacterial adherence, because it was overexpressed only in in the adhesive S. aureus (Pat36, Pat42, Pat45) and S. aureus ATCC 29213. MTT assay showed that metabolic activity was independent of the infection with S. aureus. Respiration assay indicated that metabolic activity of both SaOS2 and MG63 cell lines were stimulated after being infected with selected S. aureus isolates (Pat 9, 36 and S. aureus ATCC 29213). The relation of cell adhesion and the gene expression under biofilm condition was not closely correlated. Proteome analysis of two different osteoblast cell lines with or without infection with S. aureus showed that genes involved in adhesion were mostly not expressed in osteoblasts. Only fibronectin and collagen were detectable by mass spectrometry.
To conclude, it can be seen that neither the presence of the respective genes nor the gene products can be regarded as a sufficient predictive marker for adhesion. Gene expression analysis suggests that there may not be a uniform mechanism behind the adhesion of the examined S. aureus isolates, but that different adhesion proteins are involved. The increased adhesion is not correlated with increased invasion, which makes adhesion uninteresting as a possible therapeutic target. With respect to intracellular survival, autophagy in the host cells was turned on after infection with the S. aureus isolates tested here, indicating an onset of autophagy, which was regardless of the outcome. This study highlights a controversial role of the autophagy pathways with respect to clearance of S. aureus indicating that autophagy is necessary but not sufficient for infection. Furthermore, bacteria-induced autophagy differs from a starvation-induced one, making autophagy an interesting treatment option for the implant-associated infection. |
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| Umfang: | 92 Seiten |
| DOI: | 10.17192/z2022.0348 |