Rapid profiling of the preterm infant gut microbiota using nanopore sequencing aids pathogen diagnostics

Abstract

The Oxford Nanopore MinION sequencing platform offers direct analysis of DNA reads as they are generated, which combined with its low cost, low power and extremely compact size, makes the device attractive for in-field or clinical deployment, e.g. rapid diagnostics. We employed the MinION platform for shotgun metagenomic sequencing and analysis of mixed gut-associated microbial communities; firstly, we used a 20 species human microbiota mock community to show that Nanopore metagenomic data can be classified reliably and rapidly. Secondly, we profiled bacterial DNA isolated from faeces from preterm infants at increased risk of sepsis and necrotising enterocolitis to analyse their gut microbiota. Using longitudinal samples, and comparing Illumina to MinION, we captured the diversity of the immature gut microbiota and observed how its complexity changes over time in response to interventions, i.e. probiotic, antibiotics and episodes of suspected sepsis. Finally, we performed a 'real-time' run from sample to analysis using a faecal sample of a critically ill infant. Real-time analysis was facilitated by our new NanoOK RT software package. We determined that we can reliably identify potentially pathogenic taxa (i.e. Klebsiella pneumoniae) along with corresponding AMR gene profiles in as little as one hour, post sequencing start. Furthermore, data obtained revealed insights into how antibiotic treatment decisions may be rapidly modified in response to specific AMR profiles, which was validated using pathogen isolation, whole genome sequencing and antibiotic susceptibility testing. Our results demonstrate that MinION sequencers offer the ability to progress from clinical samples to a potential tailored patient antimicrobial treatment in just a few hours.

Genomic epidemiology of multidrug‐resistant Gram‐negative organisms

“…For example, a recent study applied metagenomic sequencing directly to prosthetic joint infection samples and demonstrated that this technique can be used to accurately diagnose this type of infection . The application of genomics directly to patient samples is particularly attractive for prosthetic joint infections, as the organisms that cause these infections can be present at very low levels and take 1–2 weeks to grow, whereas genomic pipelines can detect organisms and identify clinically relevant phenotypes within hours . The management of another slow‐growing organism for which resistance is a concern, Mycobacterium tuberculosis , is another clinical situation that might be improved with rapid genomic diagnostics.…”

“…Further illustrating the potential for rapid turnaround, Leggett et al . recently demonstrated real‐time organism and resistance profile identification from the feces of an ill infant that took less than 1 hour …”

“…When combined with future methods of improved susceptibility prediction, a promising technology in the realm of rapid identification and susceptibility testing is the Oxford Nanopore platform. Already, this platform has the capacity to generate sequence data sufficient for species identification in under an hour with computational steps performed on standard laptop computers . Whether these new technologies are best applied in everyday diagnostic workflows or reserved for surveillance and outbreak settings remains to be evaluated.…”

“…149 The application of genomics directly to patient samples is particularly attractive for prosthetic joint infections, as the organisms that cause these infections can be present at very low levels and take 1-2 weeks to grow, whereas genomic pipelines can detect organisms and identify clinically relevant phenotypes within hours. [150][151][152][153] The management of another slow-growing organism for which resistance is a concern, Mycobacterium tuberculosis, is another clinical situation that might be improved with rapid genomic diagnostics. Votintseva et al recently showed highly accurate identification and susceptibility profiling of clinical M. tuberculosis samples in ß12 h, whereas culturedependent phenotypic methods for M. tuberculosis susceptibility profiling can take weeks to months.…”

“…57,154 Further illustrating the potential for rapid turnaround, Leggett et al recently demonstrated real-time organism and resistance profile identification from the feces of an ill infant that took less than 1 hour. 152 Before an organism has been identified in a clinical diagnostic laboratory, patients with severe infections (e.g., bloodstream infections) are often treated empirically, and they are then switched to an antibiotic with known efficacy once a pathogen and its susceptibilities are determined. 155 In addition to organism identification, genomic methods have the potential to decrease time-to-appropriate-therapy initiation by identifying resistance markers and virulence factors.…”