DOI: 10.5061/dryad.7m0cfxq3r
title: Genomic DNA of clinical M. abscessus strains

description: Introduction. Mycobacterium abscessus (Mab) is a pathogenic bacterium that
 can cause severe lung infections, particularly in individuals with cystic
 fibrosis. Mab colonies can exhibit either a smooth (S) or rough (R)
 morphotype, influenced by the presence or absence of glycopeptidolipids
 (GPL) on their surface, respectively. Despite the clinical significance of
 these morphotypes, the relationship between GPL levels, morphotype, and
 the pathogenesis of Mab infections remains poorly understood. Gap
 Statement. The mechanisms and implications of GPL production and
 morphotypes in clinical Mab infections are unclear. There is a gap in
 understanding their correlation with infectivity and pathogenicity,
 particularly in patients with underlying lung disease. Aim. This study
 aimed to investigate the correlation between Mab morphology, GPL, and
 infectivity by analysing strains from cystic fibrosis patients'
 sputum samples. Methodology. Mab was isolated from patient sputum
 samples and categorised by morphotype, GPL profile, and
 replication rate in macrophages. A high-content ex vivo infection
 model using THP-1 cells assessed the infectivity of both clinical and
 laboratory strains. Results. Our findings revealed that around 50% of
 isolates displayed mixed morphologies. GPL analysis confirmed a consistent
 relationship between GPL content and morphotype was only found only in
 smooth isolates. Across morphotype groups, no differences were observed in
 vitro, yet clinical R strains were observed to replicate at higher levels
 in the THP-1 infection model. Moreover, the proportion of infected
 macrophages was notably higher among clinical R strains compared to their
 S counterparts at 72 hours post-infection. Clinical variants also infected
 THP-1 cells at significantly higher rates compared to laboratory strains,
 highlighting the limited translatability of lab strain infection data to
 clinical contexts. Conclusion. Our study confirmed the general correlation
 between morphotype and GPL levels in smooth strains yet unveiled more
 variability within morphotype groups than previously recognised,
 particularly during intracellular infection. As the rough morphotype is of
 highest clinical concern, these findings contribute to the expanding
 knowledge base surrounding Mab infections, offering insights that can
 steer diagnostic methodologies, and treatment approaches.
description: <strong>DNA extraction and
 sequencing</strong> Unseparated isolates were
 grown in 7H9OADCT on a shaker at 37 °C to an OD600 of 1.0-2.0. Genomic DNA
 was extracted with the CTAB method, eluted into 50µL of sterile water and
 stored at -20 °C. Samples were transferred to the
 Genome Sciences Centre (UBC, Vancouver, Canada) for sequencing on the
 Illumina MiSeq platform (Illumina, San Diego, CA, USA). Each genome was
 sequenced to a 100-fold depth of coverage. FASTQ files of the paired-end
 reads were inspected for overall quality using the FastQC program and
 assembled using SPAdes v.3.9.0. Assembled genomes were aligned to the
 reference strain ATCC19977/ CIP104536 S through conserved genomic regions
 with Parsnp. Core genome single nucleotide polymorphisms (SNPs) identified
 from the alignment were then used to construct a phylogenetic tree, which
 was visualised with FigTree v.1.4.4.
description: # Genomic DNA of clinical M. abscessus strains
 [https://doi.org/10.5061/dryad.7m0cfxq3r](https://doi.org/10.5061/dryad.7m0cfxq3r) Sequence data of each sample have been uploaded as forward (R1) and reverse (R2) fastq files.\ Files are labelled with the sample name and forward/reverse designation e.g., Mab10_R1.fastq.gz (SampleName_ForwardRead.fastq.gz). **DNA extraction and sequencing** Unseparated isolates were grown in 7H9OADCT on a shaker at 37 °C to an OD600 of 1.0-2.0.  Genomic DNA was extracted with the CTAB method, eluted into 50µL of sterile water and stored at -20 °C. Samples were transferred to the Genome Sciences Centre (UBC, Vancouver, Canada) for sequencing on the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Each genome was sequenced to a 100-fold depth of coverage. FASTQ files of the paired-end reads were inspected for overall quality using the FastQC program and assembled using SPAdes v.3.9.0. Assembled genomes were aligned to tthe reference strain ATCC19977/ CIP104536 S through conserved genomic regions with Parsnp. Core genome single nucleotide polymorphisms (SNPs) identified from the alignment were then used to construct a phylogenetic tree, which was visualised with FigTree v.1.4.4.