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

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

doi:10.3389/fmicb.2020.01687

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.

Highlights

Metagenomic next-generation sequencing refers to shotgun sequencing of all available DNA and/or RNA in a sample followed by the precise taxonomic identification and classification of each sequence (Couto et al, 2018; Rutanga et al, 2018)
Three different types of blood culture bottles (BCBs) samples were obtained during that study: BCBs inoculated with periprosthetic joint tissue (PJT) clinical samples from patients with suspicion of prosthetic joint infection (PJI), BCBs inoculated with tissue spiked with bacterial species reported as common microbiological causes of PJI and a negative control which was prepared by inoculating sterilized tissue from a crushed native femoral head
Since there is no standard procedure for the isolation of DNA from BCBs inoculated with PJT, we initially examined the performance of QIAamp BiOstic Bacteremia DNA Kit pretreated and untreated with the MolYsis basic5 kit

Summary

Introduction

Metagenomic next-generation sequencing (mNGS) refers to shotgun sequencing of all available DNA and/or RNA in a sample followed by the precise taxonomic identification and classification of each sequence (Couto et al, 2018; Rutanga et al, 2018). Shotgun-metagenomics has a huge potential, in areas where conventional diagnostic methods have limitations such as in prosthetic joint infection (PJI) (Thoendel et al, 2018). It is a promising approach opening huge opportunities for detecting, identifying and characterizing all potential pathogens, providing at the same time additional inputs on important characteristics for clinical management, such as antibiotic resistance determinants, virulence factors, and bacterial evolution (Wilson et al, 2019). Several reviews have summarized the advances, limitations and challenges in the field (Padmanabhan et al, 2013; Simner et al, 2018; Chiu and Miller, 2019)

Methods

Results

Conclusion