Abstract
Our culture-independent nanopore shotgun metagenomic sequencing protocol on biopsies has the potential for same-day diagnostics of orthopaedic implant-associated infections (OIAI). As OIAI are frequently caused by Staphylococcus aureus, we included S. aureus genotyping and virulence gene detection to exploit the protocol to its fullest. The aim was to evaluate S. aureus genotyping, virulence and antimicrobial resistance genes detection using the shotgun metagenomic sequencing protocol. This proof of concept study included six patients with S. aureus-associated OIAI at Akershus University Hospital, Norway. Five tissue biopsies from each patient were divided in two: (1) conventional microbiological diagnostics and genotyping, and whole genome sequencing (WGS) of S. aureus isolates; (2) shotgun metagenomic sequencing of DNA from the biopsies. Consensus sequences were analysed using spaTyper, MLST, VirulenceFinder, and ResFinder from the Center for Genomic Epidemiology (CGE). MLST was also compared using krocus. All spa-types, one CGE and four krocus MLST results matched Sanger sequencing results. Virulence gene detection matched between WGS and shotgun metagenomic sequencing. ResFinder results corresponded to resistance phenotype. S. aureus spa-typing, and identification of virulence and antimicrobial resistance genes are possible using our shotgun metagenomics protocol. MLST requires further optimization. The protocol has potential application to other species and infection types.
Highlights
The possible ramifications of orthopaedic implant-associated infections (OIAI) are salient, including reduced function, poorer treatment outcome, and increased mortality.Delayed or poorly targeted treatment can lead to selection for antimicrobial resistant bacterial strains, revision surgery, or removal of the implant device [1,2]
As OIAI are most frequently caused by Staphylococcus aureus [6], we have included S. aureus protein A-typing, multilocus sequence typing (MLST), and virulence gene detection to exploit the new protocol to its fullest
The patients are a sub-set of those included in an earlier study testing a culture-independent shotgun metagenomic sequencing protocol for rapid diagnostics of OIAI patients [5]
Summary
The possible ramifications of orthopaedic implant-associated infections (OIAI) are salient, including reduced function, poorer treatment outcome, and increased mortality.Delayed or poorly targeted treatment can lead to selection for antimicrobial resistant bacterial strains, revision surgery, or removal of the implant device [1,2]. The possible ramifications of orthopaedic implant-associated infections (OIAI) are salient, including reduced function, poorer treatment outcome, and increased mortality. The current gold standard for microbiological diagnostics of OIAI is culturing [3], which has been criticized for both its sensitivity and the time to diagnosis [4]. Culture-independent shotgun metagenomic sequencing looks to be a promising alternative in the effort to provide rapid, accurate diagnoses of OIAI. Our recently tested protocol using nanopore shotgun metagenomic sequencing directly on tissue biopsies has the potential to facilitate same-day diagnostics and administration of targeted treatment [5]. As OIAI are most frequently caused by Staphylococcus aureus [6], we have included S. aureus protein A (spa)-typing, multilocus sequence typing (MLST), and virulence gene detection to exploit the new protocol to its fullest
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