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.

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 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.