Genomics-Based Identifcation of Microorganisms in Human Ocular Body Fluid

Abstract Advances in genomics have the potential to revolutionize clinical diagnostics. Here, we examine the microbiome of vitreous (intraocular body fluid) from patients who developed endophthalmitis following cataract surgery or intravitreal injection. Endophthalmitis is an inflammation of the intraocular cavity and can lead to a permanent loss of vision. As controls, we included vitreous from endophthalmitis-negative patients, balanced salt solution used during vitrectomy and DNA extraction blanks. We compared two DNA isolation procedures and found that an ultraclean production of reagents appeared to reduce background DNA in these low microbial biomass samples. We created a curated microbial genome database (>5700 genomes) and designed a metagenomics workflow with filtering steps to reduce DNA sequences originating from: (i) human hosts, (ii) ambiguousness/contaminants in public microbial reference genomes and (iii) the environment. Our metagenomic read classification revealed in nearly all cases the same microorganism that was determined in cultivation- and mass spectrometry-based analyses. For some patients, we identified the sequence type of the microorganism and antibiotic resistance genes through analyses of whole genome sequence (WGS) assemblies of isolates and metagenomic assemblies. Together, we conclude that genomics-based analyses of human ocular body fluid specimens can provide actionable information relevant to infectious disease management

Reconstruction of ancient microbial genomes from the human gut

Loss of gut microbial diversity in industrial populations is associated with chronic diseases, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000–2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces

The molecular blueprint of a fungus by next-generation sequencing (NGS)

Sequencing the whole genome of an organism is invaluable for its comprehensive molecular characterization and has been drastically facilitated by the advent of high-throughput sequencing techniques. Especially in clinical microbiology the impact of sequenced strains increases as resistance and virulence markers can easily be detected. Here, we describe a combined approach for sequencing a fungal genome and transcriptome from initial nucleic acid isolation through the generation of ready-to-load DNA libraries for the Illumina platform and the final step of genome assembly with subsequent gene annotation

Ambiguous sequences at different cut off levels - (Item E)

For the evaluation of ambiguous sequences in microbial reference genomes we assessed different cut of levels for the generation of a curated microbial genome database. For each sequence fragment we determined the ratio between the number of negative BLAST hits (query genus ≠ subject genus) and all BLAST hits.<br><br>For a detailed description of this analysis, see the Supplemental Materials section of the publication.<br><br><b>Reference</b> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Ambiguous sequences in public microbial genomes - Sequence List - (Item D)

Scaffolds and contigs predicted to be ambiguous in the 5754 public microbial genomes examined in Kirstahler <i>et al</i>. (2017).<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Read counts for most abundant microbial agent in patient samples - (Item G)

Vitreous was analyzed using metagenomics using two DNA isolation methods (QIAamp DNA Mini Kit, QIA; QIAamp UCP Pathogen Mini kit, UCP) and the taxonomic affiliation of reads was determined using Kraken, Bracken, and BLASTn analysis. <br>The read counts for the most abundant microbial agent in endophthalmitis patients are listed. <br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

The effect of contaminated reference genomes in clinical metagenomics (Item B)

The importance of using curated microbial reference genome databases. <br> Classifying unfiltered quality trimmed reads using a Kraken database composed of non-curated microbial reference genomes and the human reference results in the identification of many reads that are mapping to <i>Toxoplasma gondii</i> and <i>Plasmodium vivax</i>. In fact, these genomes recruit more reads than the causing agent <i>Enterococcus faecalis</i>. The classification is improved when human DNA sequences are filtered out prior to classification and a cleaned reference database is used.<br>The sankey diagrams were created using Pavian (https://www.biorxiv.org/content/early/2016/10/31/084715). <br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Comparison between QIAamp DNA Mini Kit [QIA], QIAamp UCP Pathogen Mini kit [UCP] - (Item O)

Bayesian estimation analysis of reads counts obtained from samples extracted with both, QIAamp DNA Mini Kit [QIA], QIAamp UCP Pathogen Mini kit [UCP]. <br><br> The analysis was carried out according to BEST by John K. Kruschke (Journal of Experimental Psychology: General, 2013, v.142(2), pp.573-603. doi: 10.1037/a0029146).<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b> <b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p><br><br

Bacteriophages/viruses in endophthalmitis and control samples - (Item L)

Bacteriophages (DNA viruses) detected in vitreous from endophthalmitis-positive and -negative patients, basal salt solution samples, and DNA extraction controls.<b><br><br>Reference</b> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p