In vivo gene expression in a Staphylococcus aureus prosthetic joint infection characterized by RNA sequencing and metabolomics: a pilot study
Project Description
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Investigated Staphylococcus aureus gene expression and metabolism in a prosthetic joint infection with acute presentation.
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Combined deep RNA sequencing and NMR-based metabolomics to study the bacterial transcriptome and joint fluid metabolome in vivo.
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Compared in vivo findings with S. aureus grown in vitro to highlight differences in gene expression and metabolism.
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Found high expression of siderophore synthesis and virulence genes, indicating active infection.
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Identified that bacteria were sustained by amino acids, glycans, and nucleosides, not sugars or lipids.
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Detected ethanol and upregulated fermentation genes, suggesting severe oxygen limitation in the joint environment.
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Demonstrated how combined transcriptomic and metabolomic analysis can uncover mechanisms behind hard-to-diagnose prosthetic infections.
Project Details
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Successfully isolated and cultured a pure Staphylococcus aureus strain from an infected joint prosthesis using standard methods and amplicon sequencing.
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Performed whole-genome sequencing, generating 17.8 million reads, yielding a high-quality draft genome with 2562 protein-coding genes.
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Identified the isolate as spa type t908 and Clonal Complex 45 (CC45), genetically related to the USA600-BAA1754 strain.
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Conducted RNA-seq analysis comparing in vivo and in vitro conditions, with 350 million reads from infected joint fluid and 430 differentially expressed genes (317 upregulated, 113 downregulated).
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Detected significant upregulation of virulence genes in vivo, including γ-hemolysins (up to 776-fold), adhesins, and immune evasion molecules.
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Notably high expression of regulatory systems saeRS and vraSR, known to control virulence and cell wall synthesis, likely induced by β-lactam antibiotic treatment.
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Genome analysis confirmed pansusceptibility to major antibiotics, with no resistance genes detected for β-lactams, macrolides, or aminoglycosides.
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Observed increased expression of efflux pumps and cell wall biosynthesis genes, possibly as a response to antibiotic stress.
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In vivo gene expression revealed adaptation to hypoxic conditions, with strong activation of fermentation and arginine catabolism pathways (e.g., ADI pathway up to 279-fold).
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Detected upregulation of iron acquisition genes, specifically the staphyloferrin B biosynthesis operon, supporting bacterial survival in iron-limited environments.
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Revealed that free amino acids, nucleosides, and host-derived glycans were likely key nutrient sources for S. aureus in vivo.
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Contrary to expectations, most hydrolytic enzyme genes were not expressed in vivo, suggesting nutrient liberation may have been driven by host inflammatory enzymes rather than bacterial secretion.
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Collected and analyzed Staphylococcus aureus directly from infected joint aspirates of human patients.
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Successfully re-isolated the same bacterial strains for in vitro comparison.
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Performed integrated transcriptomic and metabolomic profiling of in vivo vs. in vitro bacterial states.
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Identified key differences in glucose utilization and energy metabolism in the host environment.
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Revealed suppressed expression of nitric oxide detoxification genes in vivo, suggesting immune evasion strategies.
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Detected altered amino acid uptake patterns, reflecting host-induced metabolic stress.
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Provided the first systems-level insight into how S. aureus adapts transcriptionally and metabolically during acute prosthetic joint infection.
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Validated the importance of studying pathogens in clinically relevant, in vivo-like conditions.
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