Towards diagnostic metagenomics of Campylobacter in fecal samples

Abstract

Background: The development of diagnostic metagenomics is driven by the need for universal, culture-independent methods for detection and characterization of pathogens to substitute the time-consuming, organism-specific, and often culture-based laboratory procedures for epidemiological source-tracing. Some of the challenges in diagnostic metagenomics are, that it requires a great next-generation sequencing depth and unautomated data analysis.

Results: DNA from human fecal samples spiked with 7.75 × 10¹-7.75 × 10⁷ colony forming unit (CFU)/ml Campylobacter jejuni and chicken fecal samples spiked with 1 × 10²-1 × 10⁶ CFU/g Campylobacter jejuni was sequenced and data analysis was done by the metagenomic tools Kraken and CLARK. More hits were obtained at higher spiking levels, however with no significant linear correlations (human samples p = 0.12, chicken samples p = 0.10). Therefore, no definite detection limit could be determined, but the lowest spiking levels found positive were 7.75 × 10⁴ CFU/ml in human feces and 10³ CFU/g in chicken feces. Eight human clinical fecal samples with estimated Campylobacter infection loads from 9.2 × 10⁴-1.0 × 10⁹ CFU/ml were analyzed using the same methods. It was possible to detect Campylobacter in all the clinical samples.

Conclusions: Sensitivity in diagnostic metagenomics is improving and has reached a clinically relevant level. There are still challenges to overcome before real-time diagnostic metagenomics can replace quantitative polymerase chain reaction (qPCR) or culture-based surveillance and diagnostics, but it is a promising new technology.

Keywords: Culture independent; Molecular typing; Next generation sequencing.

Fig. 1

Study design. Flowchart showing workflow in the study and differences between the analysis of human and chicken samples

Fig. 2

Rarefaction curves of the mean species richness for each subsample. Horizontal axes hold individuals in subsample and vertical axes hold mean species richness. a human clinical fecal samples, b human spiked fecal samples, c chicken spiked fecal samples

Fig. 3

Total bacterial composition. Human samples above, chicken samples below. Left barcharts show the composition of all reads assigned to phylum level. Right barcharts show the composition including unassigned reads. NC = negative control