Delineating Mycobacterium abscessus population structure and transmission employing high-resolution core genome multilocus sequence typing

Abstract

Mycobacterium abscessus is an emerging multidrug-resistant non-tuberculous mycobacterium that causes a wide spectrum of infections and has caused several local outbreaks worldwide. To facilitate standardized prospective molecular surveillance, we established a novel core genome multilocus sequence typing (cgMLST) scheme. Whole genome sequencing data of 1991 isolates were employed to validate the scheme, re-analyze global population structure and set genetic distance thresholds for cluster detection and taxonomic identification. We confirmed and amended the nomenclature of the main dominant circulating clones and found that these also correlate well with traditional 7-loci MLST. Dominant circulating clones could be linked to a corresponding reference genome with less than 250 alleles while 99% of pairwise comparisons between epidemiologically linked isolates were below 25 alleles and 90% below 10 alleles. These thresholds can be used to guide further epidemiological investigations. Overall, the scheme will help to unravel the apparent global spread of certain clonal complexes and as yet undiscovered transmission routes.

Fig. 1. CgMLST-based neighbor-joining tree comprising 1786 Mycobacterium abscessus isolates.

1. Strains that were previously classified into one of seven dominant circulating clones (DCC) based on core genome single nucleotide polymorphism (cgSNP)/FastBAPS analysis. DCC clades (inner circle) are delineated and colored based on the most recent common ancestral node comprising all strains with known DCC classification. 2. Sequence types (ST) according to the nomenclature of the 7-loci MLST scheme hosted on pubMLST. 3. Mash distance-based subspecies classification.

Fig. 2. CgMLST-based minimum spanning tree visualizing the genetic relationship between isolates from three extra-pulmonary outbreaks and 12 putative pulmonary transmission clusters.

Isolates were previously clustered in six independent studies^{, , , –}. Node labels correspond to isolate ID. The size of the nodes is proportional to the number of samples with the same cgMLST profile (i.e. with allelic distance=0). Distances (SNPs in red and alleles in black) between two isolates are displayed on the branches (not to scale). Dashed boxes indicate patients that have attended the same cystic fibrosis (CF) healthcare center at the same time (i.e. putative patient-to-patient transmission event). OM: otitis media.

Fig. 3. Boxplots of pairwise genetic distances (i.e. cgMLST allele differences) between related isolates.

A Pairwise distances between isolates from previously reported extra-pulmonary Mab outbreaks^, (n = 444 pairwise comparisons). B Pairwise distances between pulmonary isolates from cystic fibrosis (CF) patients for which epi data suggested nosocomial transmission^{, ,} (n = 43 pairwise comparisons). C Pairwise distances between pulmonary isolates from predominantly cystic fibrosis (CF) patients for which epi data did not support nosocomial transmission^, (n = 23 pairwise comparisons). For B and C, clusters were previously defined with a threshold of 30 SNPs. D Pairwise distances between isolates from the same patient^{, ,} (n = 1,150 pairwise comparisons). All outliers are related to patient 5. Dashed lines indicate tentative thresholds to initiate further epidemiological investigations. Solid lines indicate median pairwise allele distance, box represent interquartile range (IQR), whiskers extend to 1.5 of the IQR, dots represent outliers. Links are the amount of pairwise comparisons included per group. Pairwise allele distances between groups were compared with a Wilcoxon rank sum test (with Bonferroni correction): *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. CgMLST-based minimum spanning tree visualizing the genetic relationship between sequential isolates from the same patient (within-patient diversity).

Patients from two studies^, with more than three isolates are displayed. Node labels correspond to the date of Mycobacterium abscessus isolate collection. The size of the nodes is proportional to the number of samples with identical cgMLST profile (i.e. with distance=0). Allelic distances between two isolates are displayed on the branches (not to scale).