Metagenomic nanopore sequencing is demonstrating potential as a tool for diagnosis of infections directly from clinical samples. We have previously shown nanopore sequencing can be used to determine the causative bacterial species in prosthetic joint infections (PJI). However, to make predictions regarding antimicrobial resistance, human DNA contamination must be reduced so a greater proportion of sequence data corresponds to the microbial portion of the DNA extract. Here, we utilise selective DNA extraction from sonication fluid samples to begin to make predictions regarding antimicrobial resistance in PJI.
We investigated host cell DNA depletion with 5% saponin selective human cell lysis followed by nuclease digestion. Subsequently, bacterial cells were mechanically lysed before DNA extraction. Sequencing libraries from samples treated with and without saponin were prepared with a Rapid PCR Barcoding Kit1 and sequenced in multiplexes of 2–8 samples/flowcell on a GridION. Sequencing reads were analysed using the CRuMPIT pipeline and thresholds to indicate presence of a specific bacterial genus/species were investigated. Antimicrobial resistance determinants were detected using previously published sequences specifically for Staphylococcus aureus, as an example organism frequently causing PJI.
247 DNA extracts from 115 sonication fluids plus controls were subjected to metagenomic sequencing, comprising extracts from 67 culture-positive (10 of which were culture-positive at <50 CFU/ml) and 48 culture-negative samples. 5% saponin depleted human DNA contamination, reducing the number of human sequenced bases to a median 12% from 98% in comparison to 5μm filtration without saponin. In 11 samples 5% saponin depleted human bases by <12% in comparison to 5μm filtration, which may be indicative of incomplete depletion. Bacteria observed in sonication fluid culture were identified to species-level in 49/65 (75%) cases, and to genus-level in 51/65 (78%). Specificity of sequencing was 103/114 (90%). Sequencing made a completely successful prediction of antimicrobial susceptibility in 8/19 S. aureus culture-positive samples treated with 5% saponin, and a partial prediction in 5/19 for the 8 antibiotics investigated. Without 5% saponin treatment sequencing could only detect a limited number of AMR determinants in 3/19 samples. Sequencing correctly predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient sequence data were available.
Nanopore metagenomic sequencing can provide species identification in PJI. Additionally, depletion of human DNA improves depth of coverage and allows detection of antimicrobial resistance determinants, demonstrating as a proof of principle that nanopore sequencing could potentially provide a complete diagnostic tool in PJI.
1Oxford Nanopore Technologies