Draft Genome Sequence of a Mycobacterium Strain Isolated from a Clinical Wound Sample

ABSTRACT We report the draft genome sequence of an unusual Mycobacterium isolate recovered from a patient’s arm tissue. The 4,025,753-bp draft genome exhibits a GC content of 71.02%, and a 16S rRNA gene analysis found that the closest relative was Mycobacterium grossiae.

M ycobacteria are aerobic, acid-fast, nonmotile, non-spore-forming bacilli, and some species, such as Mycobacterium tuberculosis, are important human pathogens. Mycobacterium other than tuberculosis (MOTTs), also called nontuberculous mycobacteria (NTMs), can also cause human disease. Recent changes to mycobacterial taxonomy include the subgenera Mycolicibacterium, Mycolicibacter, Mycolicibacillus, and Mycobacteroides (1); these contain over 180 species and are rapidly expanding.
A Mycobacterium-like isolate was recovered from arm tissue collected during surgical amputation following a motor vehicle accident. The isolate was detected after a 38-day incubation at 35°C in a Mycobacteria Growth Indicator Tube (MGIT) (Becton Dickinson). Clinical identification methods, including Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, failed to provide a genus-or species-level identification (2). However, the isolate was acid fast and a scotochromogen (3). Subcultured growth on Middlebrook 7H10 agar in atmospheric air was observed at 30°C and 35°C after 3 days (rapid grower). The isolate was forwarded to the University of Texas at Tyler mycobacteriology laboratory for susceptibility testing and was pansusceptible at the lowest dilutions tested (Table 1) (4).
To characterize this isolate, it was cultured for whole-genome sequencing on Middlebrook 7H10 mycobacterial agar for 14 days at 30°C in O 2 . Colony growth was suspended in 1 mL of molecular biology-grade water. DNA was purified using the QIAamp BiOstic bacteremia DNA kit (Qiagen), followed by use of the Nextera XT library preparation kit (Illumina), and was sequenced using the NovaSeq 6000 sequencing system to acquire 2 Â 150-bp pairedend reads. For all software used subsequently, default parameters were used unless otherwise specified. We generated 8,960,765 reads after quality filtering using Trimmomatic v0.36 (5). SPAdes v3.13.0 (6) was used for de novo assembly of a draft genome, and assembly quality was measured by QUAST (7). The draft genome was 4,025,753 bp, with a GC content of 71.02%, and consisted of 23 contigs, with an N 50 value of 401,536 bp and coverage of 333Â. Contigs of #500 bp were removed before assembly deposition. The genome was annotated with PGAP (8) and contained 3,946 coding sequences.
Using RNAmmer v1.2 (9), we isolated 16S rRNA gene sequences from the draft genome and 17 other actinomycete taxa for comparison, with pairwise identity determined using the EZBioCloud database (10). This analysis confirmed that the closest relative was Mycobacterium grossiae, with other species of the subgenus Mycolicibacterium having .98% identity. We aligned the corresponding gene sequences using MUSCLE and constructed an approximate maximum likelihood tree using FastTree (11,12). Phylogenetic analysis of 16S rRNA gene alignment revealed that this Mycobacterium genomospecies and M. grossiae formed a clade distinct from other well-known representative species of the four mycobacterial subgenera (Fig. 1). Previous phylogenetic analyses of NTMs clarified that M. grossiae grouped with the fast-growing and primarily environmental mycobacterial subgenus Mycolicibacterium (1), although M. grossiae remains unclassified by subgenus. Given the phenotypic characteristics of this Mycobacterium genomospecies and its genotypic attributes, it may represent a missing link between the Mycobacterium and Mycolicibacterium subgenera.
This study was performed with institutional review board (IRB) approval from Washington University in St. Louis (IRB approval number 202204102).  Data availability. This whole-genome shotgun project for Mycobacterium sp. strain MYCO198283 has been deposited in GenBank under DDBJ/ENA/GenBank accession number JAJQJI000000000, BioProject accession number PRJNA759261, BioSample accession number SAMN21161762, and SRA accession number SRS11245207.