Shotgun-Metagenomics on Positive Blood Culture Bottles Inoculated With Prosthetic Joint Tissue: A Proof of Concept Study

Adriana Sanabria; Erik Hjerde; Mona Johannessen; Johanna Ericson Sollid; Gunnar Skov Simonsen; Anne-Merethe Hanssen

doi:10.3389/fmicb.2020.01687

Shotgun-Metagenomics on Positive Blood Culture Bottles Inoculated With Prosthetic Joint Tissue: A Proof of Concept Study

Front Microbiol. 2020 Jul 17:11:1687. doi: 10.3389/fmicb.2020.01687. eCollection 2020.

Authors

Adriana Sanabria¹, Erik Hjerde², Mona Johannessen¹, Johanna Ericson Sollid¹, Gunnar Skov Simonsen^{1

3}, Anne-Merethe Hanssen¹

Affiliations

¹ Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT - The Arctic University of Norway, Tromsø, Norway.
² Department of Chemistry, Centre for Bioinformatics, UiT - The Arctic University of Norway, Tromsø, Norway.
³ Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway.

Abstract

Clinical metagenomics is actively moving from research to clinical laboratories. It has the potential to change the microbial diagnosis of infectious diseases, especially when detection and identification of pathogens can be challenging, such as in prosthetic joint infection (PJI). The application of metagenomic sequencing to periprosthetic joint tissue (PJT) specimens is often challenged by low bacterial load in addition to high level of inhibitor and contaminant host DNA, limiting pathogen recovery. Shotgun-metagenomics (SMg) performed directly on positive blood culture bottles (BCBs) inoculated with PJT may be a convenient approach to overcome these obstacles. The aim was to test if it is possible to perform SMg on PJT inoculated into BCBs for pathogen identification in PJI diagnosis. Our study was conducted as a laboratory method development. For this purpose, spiked samples (positive controls), negative control and clinical tissue samples (positive BCBs) were included to get a comprehensive overview. We developed a method for preparation of bacterial DNA directly from PJT inoculated in BCBs. Samples were processed using MolYsis5 kit for removal of human DNA and DNA extracted with BiOstic kit. High DNA quantity/quality was obtained, and no inhibition was observed during the library preparation, allowing further sequencing process. DNA sequencing reads obtained from the BCBs, presented a low proportion of human reads (<1%) improving the sensitivity of bacterial detection. We detected a 19-fold increase in the number of reads mapping to human in a sample untreated with MolYsis5. Taxonomic classification of clinical samples identified a median of 96.08% (IQR, 93.85-97.07%; range 85.7-98.6%) bacterial reads. Shotgun-metagenomics results were consistent with the results from a conventional BCB culture method, validating our approach. Overall, we demonstrated a proof of concept that it is possible to perform SMg directly on BCBs inoculated with PJT, with potential of pathogen identification in PJI diagnosis. We consider this a first step in research efforts needed to face the challenges presented in PJI diagnoses.

Keywords: blood culture bottles; clinical metagenomics; prosthetic joint infection; prosthetic joint tissue; shotgun-metagenomics.