title: Rapid whole-genome sequencing for detection and characterization of microorganisms directly from clinical samples.
abstract: Whole-genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples, this could further reduce diagnostic times and thereby improve control and treatment. A major bottleneck is the availability of fast and reliable bioinformatic tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatic tools for the analysis of sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria, and direct sequencing on pellets from the urine samples. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples but in pure cultures from only 17 samples. WGS improved the identification of the cultivated bacteria, and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibilities. Complete agreement was observed between species identification, multilocus sequence typing, and phylogenetic relationships for Escherichia coli and Enterococcus faecalis isolates when the results of WGS of cultured isolates and urine samples were directly compared. Sequencing directly from the urine enabled bacterial identification in polymicrobial samples. Additional putative pathogenic strains were observed in some culture-negative samples. WGS directly on clinical samples can provide clinically relevant information and drastically reduce diagnostic times. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem, a publicly available bioinformatic tool was developed in this study.
authors.affiliation: National Food Institute, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Hasman
authors.forename: Dhany
authors.initials: D
authors.lastname: Saputra
authors.forename: Thomas
authors.initials: T
authors.lastname: Sicheritz-Ponten
authors.forename: Ole
authors.initials: O
authors.lastname: Lund
authors.forename: Christina Aaby
authors.initials: CA
authors.lastname: Svendsen
authors.forename: Niels
authors.initials: N
authors.lastname: Frimodt-Møller
authors.forename: Frank M
authors.initials: FM
authors.lastname: Aarestrup
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D001431:Bacteriological Techniques / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D014552:Urinary Tract Infections / Q000175:diagnosis / Q000382:microbiology; D014556:Urine / Q000382:microbiology