Abstract
The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus, Corynebacterium jeikeium and Rothia dentocariosa, the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.
Highlights
Sample culturing is the main method used to identify pathogens in clinical microbiology
We eventually looked for genes that are involved in antibiotic resistance to link, if possible, antibiotic susceptibility testing (AST) for M. abscessus and C. jeikeium performed by the bacteriology laboratory (Supplementary Table S1) to our metagenomic data
847 reads were aligned to ResFinder database, most of which (94.9%) mapped to genes associated with resistance to macrolide antibiotics (Figure 3A; Table 4). We investigated whether those Antibiotic Resistance Determinants (ARDs) came from M. abscessus and C. jeikeium either by mapping reads against reference genomes with USEARCH [20] or by retrospectively looking to the taxonomic read assignments performed by CLAssifier based on Reduced K-mers (CLARK) and Kraken
Summary
Sample culturing is the main method used to identify pathogens in clinical microbiology. It requires bacterial growth on different media as well as various atmospheric and temperature adjustments. Culture-independent screening conducted with whole-metagenome shotgun (WMGS) only needs a small amount of DNA directly taken from the sample and a bioinformatics tool which identifies bacteria by linking sequencing reads to a curated reference genome (or marker) database. The availability of whole-genome sequences and taxonomic markers from microorganisms, including yet-uncultivable ones, makes it possible, with metagenomics, to provide a more comprehensive overview of the whole microbiota. WMGS can be applied to different types of samples obtained by non-invasive (e.g., urine [1,2,3]) or invasive (e.g., bone [4]) techniques
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