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Whole-Genome Sequencing of Mycobacterium tilburgii Strain MEPHI

Abstract : Mycobacterium tilburgii is a fastidious mycobacterium which has previously been reported to cause severe disseminated infections. Genome sequencing of the M. tilburgii MEPHI clinical isolate yielded 3.14 Mb, with 66.3% GC content, and confirmed phylogenetic placement within the Mycobacterium simiae complex. O nly 13 cases of Mycobacterium tilburgii infection have been reported, highlighting host immunosuppression as a risk factor for M. tilburgii infection (1-3). M. tilburgii was never previously isolated in culture; its detection relied on the direct sequencing of internal transcribed spacer 1 (ITS1), hsp65, rpoB, and 16S rRNA genes from clinical material (2). We isolated M. tilburgii from a bone marrow sample by using a shell vial assay, as previously described (4, 5) (Fig. 1A). This organism had been previously detected in this immunocompromised patient in a biopsy specimen taken from a duodenal plaque by direct 16S rRNA gene sequencing. DNA (0.2 g/l) extracted using InstaGene matrix (Bio-Rad, Marnes-la-Coquette, France) was sequenced using MiSeq platform (Illumina, Inc., San Diego, CA, USA) paired-end sequencing and automated cluster generation with dual-indexed 2 ϫ 250-bp reads. A 535,000/mm 2 cluster density filter and a 96.3% cluster passing quality control filter were applied. A total of 8,690,521 reads were assembled using SPAdes version 3.12.0 with the option "-careful" in order to reduce the number of mismatches and short indels (6). Default parameters for k values, i.e., k-mer values of 127, 99, 77, 55, 33, and 21, were applied. A total of 977 human DNA contigs were removed via a BLASTn script that connected remotely to the NCBI database (nr/nt) (BLASTϩ 2.3.0). Annotation using Prokka version 1.13 (7) of the M. tilburgii strain MEPHI genome sequence (mean coverage depth, 6.5ϫ) comprises 102 contigs, with a total assembly size of 3,238,527 bp and 66.3% GC content (N 50 value, 56,170 bp; coverage, 5.6ϫ). One 19,595-bp contig (62.9 GC% content), exhibited 77% nucleotide similarity with Mycobacterium sp. strain KMS plasmid pMKMS02 (GenBank accession no. CP000520) using BLASTn. The M. tilburgii strain MEPHI genome was predicted to contain 4,207 genes, including 4,154 protein-coding genes, and 53 RNAs (49 tRNAs, 3 rRNAs, and one transfer-messenger RNA [tmRNA]). Phylogenetic trees based on the 3,504-bp rpoB and the 1,523-bp 16S RNA genes confirmed phylogenetic placement into the Mycobacterium simiae complex (Fig. 1B and C). The genomic similarities estimated using OrthoANI version 0.93.1 (8) and in silico DNA-DNA hybrid-ization estimated using the GGDC version 2.0 online tool (9) were 87.17% and 33.8% with M. simiae (GenBank assembly accession no. GCA_000455305), 87.15% and 33.6% with Mycobacterium sherrisii (GenBank assembly accession no. GCA_001722325), 82.88% and 26.8% with Mycobacterium triplex (GenBank assembly accession no. GCA_000689255), 82.76% and 26.6% with Mycobacterium florentinum (NCBI RefSeq accession no. NZ_LQOV00000000), 82.62% and 26.1% with Mycobacterium genavense
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Jamal Saad, Michel Drancourt, Margaret Hannan, Patrick Stapleton, Simon Grandjean Lapierre. Whole-Genome Sequencing of Mycobacterium tilburgii Strain MEPHI. Microbiology Resource Announcements, American Society for Microbiology, 2019, 8 (40), pp.933 - 952. ⟨10.1128/MRA.00933-19⟩. ⟨hal-02466356⟩

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