S LEO¹, N GAIA¹, V LAZAREVIC¹, E RUPPE¹, J SCHRENZEL<sup>1

1</sup>Genomic Research Lab, Service of Infectious Diseases, Geneva University Hospitals, Geneva, Switzerland

We report the metagenomic analyses of Illumina shot-gun sequencing libraries prepared from the broncho-alveolar lavage (BAL) of a severely immunocompromised patient developing a nosocomial pneumonia. To enrich for microbial sequences, aliquots of the BAL sample have been separately treated with two methyl-directed restriction enzymes (GlaI and McrBC) for human DNA depletion prior to sequencing. Illumina paired reads were processed, mapped to reference genomes with the two classifiers, Clark (1) and Kraken (2) and the results were correlated with culture and molecular data obtained by routine diagnostic procedures performed by the bacteriology laboratory of Geneva University Hospitals. In accordance with culture data, in the untreated aliquot the two reported bacterial pathogens Mycobacterium abscessus and Corynebacterium jeikeium as well as filamentous fungi were the most abundant organisms identified. McrBC and GlaI treatments decreased the number of human-mapped reads by 10% and 20% respectively, but with a comparable increment of unclassified reads. Most microbial sequences mapped to Burkholderia pseudomallei and Torque teno viruses, undetected by culture analyses, while the read counts for C. jeikeium and, to a lesser extent, for M. abscessus were dramatically reduced, probably because of an extensive enzymatic digestion of the samples. Therefore, further optimization of these procedures for human DNA removal is required. Overall, we report here a metagenomic approach with its bioinformatic pipeline as a possible tool for faster identification of micro-organisms in a BAL, an achievement that further supports the development of culture-independent methods in clinical routine diagnosis.


References:
1. R Ounit, S Wanamaker, T J Close, S Lonardi. BMC Genomics 2015; 16(1): 236.
2. D E Wood, S L Salzberg. Genome Biol. 2014; 15(3): R46.