Patient Characteristics
Three pairs of patients with NTM-PD who had been co-habiting for at least 15 years were enrolled (Table 1). A mother and a daughter (Patients A and B) with M. avium PD had lived in an apartment in an urban area (HOME-1) for 15 years. A couple (Patients C and D) with M. intracellulare PD had lived in a house in a rural area (HOME-2) for 30 years. A second couple (Patients E and F) with M. intracellulare PD and M. abscessus subsp. massiliense PD had lived in an apartment in an urban area (HOME-3) for 30 years. No patients were suspected of any immunodeficiency disorders. They were HIV-negative, were not taking any immunosuppressants and had no history of recurrent infection of any organs.
NTM Isolation and Sequencing
Among 12 environmental specimens from HOME-1, seven specimens from either the kitchen or the bathroom were culture-positive. Subsequently, 18 morphologically distinct isolates were purified (Supplementary Table 1, Additional File 1). However, none of the 15 environmental specimens from HOME-2 yielded any NTM isolates and only one of 15 specimens from HOME-3 yielded NTM isolates.
Table 1. Characteristics of the three patient pairs in this study
|
Age
/Relationship
|
Sources
|
NTM Collection Date
|
NTM Species by conventional method
|
Habitat
|
Radiologic findings
|
Patient A
|
81
/Mother
|
Sputum
|
5 June 2017
|
M. avium
|
HOME-1
Apartment (urban area) for 15 years.
|
Bronchiectasis and centrilobular nodules in RML/LUL lingular segments
|
Patient B
|
51
/Daughter
|
Sputum
|
19 July 2017
|
M. avium
|
Centrilobular nodules with branching opacity in RUL/RML/LUL lingula segments
|
Patient C
|
77
/Husband
|
Sputum
|
13 April 2017
|
M. intracellulare
|
HOME-2
House (rural area) for 30 years with high soil environment
|
Lung nodule in RUL
|
Patient D
|
71
/Wife
|
Sputum
|
3 May 2017
|
M. intracellulare
|
Multiple branching opacity and centrilobular nodules in both lung
|
Patient E
|
62
/Husband
|
Bronchial washing
|
29 September 2017
|
M. intracellulare
|
HOME-3
Apartment (urban area) for 30 years.
|
Bronchiectasis with peribonchial infiltration in LLL
|
Patient F
|
61
/Wife
|
Bronchial washing
|
29 September 2017
|
M. abscessus subsp. massiliense
|
Patchy opacity and nodules in RML
|
RUL; right upper lobe, RML: right middle lobe, LUL: left upper lobe, LLL: left lower lobe
On average, 19.8 million sequencing reads were obtained for each isolate (See Supplementary Table 2, Additional File 1). According to k-mer based taxonomic classification of NGS reads, 12 isolates from environmental specimens in HOME-1, isolates from Patient A and Patient B were identified as M. avium subsp. hominissuis. Isolates from Patients C, D, and E were identified as M. intracellulare, and one isolate from Patient F was identified as M. abscessus. The identifications of patient-derived isolates using NGS produced the same results as conventional PCR and direct sequencing. Two isolates from environmental specimens in HOME-1 were identified as M. fortuitum, while the remaining four isolates from HOME-1 and three isolates from HOME-3 were classified as non-NTM and were excluded.
The mean sequencing depth of identified isolates was 313× (210–487×). To reduce the potential impact of recombination and mobile genetic elements on our results, we defined core regions for each species as common sequences observed across the 17 M. avium subsp. hominissuis and 3 M. intracellulare genomes analyzed in this study (as well as 64 and 14 publicly available genomes, respectively). The core region of M. avium subsp. homonissuis consisted of 4.30 Mbp of the 5.15 Mbp genome (83%), and that of M. intracellulare consisted of 4.23 Mbp of the 5.40 Mbp genome (78%). On average, 38,377 (27,469–43,720) high confidence SNPs were detected in M. avium subsp. hominissuis and 16,464 (15–26,740) high confidence SNPs were detected in M. intracellulare within these regions (Supplementary Table 2, Additional File 1). Based on the distributions of SNP allele fraction (Supplementary Table 2, Additional File 1), all isolates were monoclonal except for the isolate from Patient C, which consisted of two clones at an 8:2 ratio (Supplementary Figure 1). The 25,441 and 2,485 high confidence SNPs from Patient C were classified as belonging to the Cmajor and Cminor clones based on SNP allele fractions.
Pairwise SNP Distances and Phylogenetic Analysis
A total of 104,531 and 102,281 genomic positions with high-confidence SNPs were identified in M. avium subsp. hominissuis and M intracellulare genome, respectively, and used for further analysis. Pairwise SNP distances between every pair of isolates at those positions were calculated and clusters on the histogram of SNP distances were observed (Figure 1). The SNP distance between the isolates from Patient A and B was 14,768. By contrast, the SNP distances among three replicates (isolates from Patient A, Kitchen Sink Faucet 1, Kitchen Sink Cold Water 3) were less than 100. (Figure 1a) Using high-confidence SNPs from 81 M. avium subsp. hominissuis genomes, phylogenetic analysis was performed (Figure 2a). The isolate from Patient A and its replicates clustered with the specimens from the kitchen (scale on surface of kitchen faucet), while the isolate from Patient B clustered with the isolates from the bathroom (hot water from bathroom faucet, hot water from showerhead) and the kitchen (cold water from kitchen faucet).
In HOME-2, the SNP distance between the M. intracellulare isolates from Patients Cmajor and D was 29,873, and the distance between Patient Cminor and D was 12,670. Those distances were even higher than the SNP distances from Patient E in HOME-3. (Figure 1b) Phylogenetic analysis with 17 M. intracellulare genomes confirmed that all three isolates from Patient C, D, and E were not closely related each other. (Figure 2b) In HOME-3, different species of NTM were isolated (M. intracellulare from Patient E and M. abscessus subsp. massiliense from patient F), and the SNP distance was not calculated.