Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing.
The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar.



Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes.



MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified.



MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.
Norwich Medical School, University of East Anglia, Norwich, UK.
K
K
Schmidt
Norwich Medical School, University of East Anglia, Norwich, UK.
S
S
Mwaigwisya
SequenceAnalysis.co.uk, Norwich Research Park, Norwich, UK.
L C
LC
Crossman
AMRHAI Reference Unit, National Infection Service, Public Health England, London, UK.
M
M
Doumith
Microbiology Department, Norfolk and Norwich University Hospital, Norwich, UK.
D
D
Munroe
Brunel University London, Uxbridge, UK.
C
C
Pires
Brunel University London, Uxbridge, UK.
A M
AM
Khan
AMRHAI Reference Unit, National Infection Service, Public Health England, London, UK.
N
N
Woodford
Brunel University London, Uxbridge, UK.
N J
NJ
Saunders
Norwich Medical School, University of East Anglia, Norwich, UK.
J
J
Wain
Norwich Medical School, University of East Anglia, Norwich, UK justin.ogrady@uea.ac.uk.
J
J
O'Grady
Norwich Medical School, University of East Anglia, Norwich, UK.|AMRHAI Reference Unit, National Infection Service, Public Health England, London, UK.
D M
DM
Livermore
D001419:Bacteria / Q000187:drug effects / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D019295:Computational Biology / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D056186:Metagenomics / Q000379:methods*; D008826:Microbial Sensitivity Tests / Q000379:methods*; D058608:Nanopores; D013997:Time Factors; D014552:Urinary Tract Infections / Q000175:diagnosis* / Q000382:microbiology; D014556:Urine / Q000382:microbiology*