Whole genome sequencing of Nontuberculous Mycobacterium (NTM) isolates from sputum specimens of cohabiting patients with NTM pulmonary disease and NTM isolates from their environment

- Whole genome sequencing of.
- Nontuberculous Mycobacterium (NTM) isolates from sputum specimens of co- habiting patients with NTM pulmonary disease and NTM isolates from their environment.
- However, recent studies using next-generation sequencing (NGS) have reported trans- continental spread of Mycobacterium abscessus among patients with cystic fibrosis..
- Results: We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co- habiting patients with NTM-PD who were suspected of patient-to-patient transmission.
- Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M.
- intracellulare and the other of whom was infected with M.
- Whole genome sequencing was performed using patients ’ NTM isolates as well as environmental specimens.
- By comparison with the genetic distances among 78 publicly available NTM genomes, NTM isolates derived from the two pairs of patients infected with the same NTM species were not closely related to each other.
- In phylogenetic analysis, the NTM isolates from patients with M.
- avium -PD clustered with isolates from different environmental sources..
- Conclusions: In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to- patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal..
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- Several explanations for this epidemio- logical change have been proposed, including awareness and improved detection of NTM-PD, increased populations with risk factors such as bronchiectasis or use of immunosuppressants, and disinfection of drinking water in urban areas resulting in selective advantages for NTM [1]..
- Since NTM are ubiquitous microorganism, it is gener- ally assumed that patients with NTM-PD acquire NTM from their environment, not from other infected individ- uals.
- However, recent NGS studies showed that this might not be the case, at least for patients with cystic fi- brosis [8, 9].
- Bryant and colleagues collected Mycobac- terium abscessus isolates from patients with cystic fibrosis, performed WGS, and analysed phylogenetic re- lationships among these isolates.
- abscessus among patients with cystic fibrosis, and identified some M.
- Although Harris and col- leagues reported no evidence of patient-to-patient trans- mission in their cohort of pediatric cystic fibrosis patients [10], NGS studies of NTM transmission have.
- This would be espe- cially important for hospital infection control, since iso- lation practices for NTM-PD patients without cystic fibrosis are not as strict as those for pulmonary tubercu- losis patients generally..
- Recently, we diagnosed three pairs of NTM-PD patients with no immunodeficiency who had been co- habiting for at least 15 years.
- We investigated the ge- nomes of NTM isolates derived from the patients and environmental samples in their houses to understand the source of infection using WGS..
- Three pairs of patients with NTM-PD who had been co- habiting for at least 15 years were enrolled (Table 1).
- Table 1 Characteristics of the three patient pairs in this study Age.
- However, none of the 15 en- vironmental specimens from HOME-2 yielded any NTM isolates and only one of 15 specimens from HOME-3 yielded NTM isolates..
- According to k-mer based taxonomic classification of NGS reads, 12 isolates from environ- mental specimens in HOME-1, isolates from Patient A and Patient B were identified as M.
- Isolates from Patients C, D, and E were identified as M.
- Two isolates from environmental specimens in HOME-1 were identi- fied 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..
- homo- nissuis consisted of 4.30 Mbp of the 5.15 Mbp genome (83.
- intracellulare consisted of 4.23 Mbp of the 5.40 Mbp genome (78.
- On average high confidence SNPs were detected in M.
- high confidence SNPs were detected in M.
- The 25,441 and 2485 high confidence SNPs from Patient C were classified as be- longing to the Cmajor and Cminor clones based on SNP allele fractions..
- The SNP distance between the isolates from Patient A and B was 14,768..
- 1a) Using high-confidence SNPs from 81 M.
- The isolate from Patient A and its rep- licates 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 bath- room (hot water from bathroom faucet, hot water from showerhead) and the kitchen (cold water from kitchen faucet)..
- intracellulare isolates from Patients C and D living together in HOME-2 (red arrow) was higher than the distance between isolates from Patients C and E living in other houses (blue arrow).
- In HOME-2, the SNP distance between the M.
- intra- cellulare 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.
- confirmed that all three isolates from Patient C, D, and E were not closely related each other.
- 2b) In HOME-3, different species of NTM were isolated (M..
- intracellulare from Patient E and M.
- massiliense from patient F), and the SNP distance was not calculated..
- (a) Isolates from Patients A and B (blue) clustered apart from environmental isolates from HOME-1, and 64 publicly available M.
- abscessus among cystic fibrosis patients [8, 9].
- The British Thoracic Society [12], US Cystic Fibrosis Foundation and European Cystic Fi- brosis Society [13] recommended that infection control policies minimize risks of patient-to-patient transmission for patients with both cystic fibrosis and M.
- However, these guidelines were only designed for patients with underlying risk factors and contain no statements for patients without immunodeficiencies.
- Among the three pairs of co-habiting NTM-PD pa- tients without overt immunodeficiency examined here, no patient pairs had near-identical NTM isolates (Fig..
- avium-PD isolates that were genetically distinct but similar to sep- arate isolates from environmental specimens in their homes.
- Although we could not identify environmental sources for the remaining two pairs of patients, one pa- tient pair was also infected with genetically distinct M..
- and the last pair of patients was infected by different species of NTM (M.
- One report showed that samples from 17 (46%) of 37 households yielded the same species of NTM found in the patient..
- Recently, variable number tandem repeat genotyping and WGS revealed that 11 of 21 (52%) patients with M.
- Patient A seemed to have ac- quired NTM from the kitchen, while Patient B acquired NTM from the bathroom or kitchen..
- A previous study reported that NTM species could be isolated from 22 of 37 (59%) households of NTM-PD patients.
- addition, a positive correlation was observed between the number of samples collected per house and the number of NTM-positive samples [15].
- this number is equal to or larger than that of most studies, suggesting that there may be sources of NTM-PD other than water..
- The patients living in HOME-2 (Patient C and D) were farmers and their home was a high soil environment that included a yard and hay (Table 1).
- The sequencing reads of NTM isolates from Patients E and F aligned to NTM reference genomes, but those from en- vironmental specimen isolates did not.
- Thus, these en- vironmental isolates were not closely related to NTM in HOME-3.
- intracellulare-PD living in low soil environments, no patients had genetically identical isolates compared with soil sample isolates from their houses [17].
- The lag time between the acquisition of NTM from the environment and diagnosis of NTM-PD also makes it more difficult to identify the source of NTM [14]..
- NTM isolates derived from three pairs of co-habiting NTM-PD patients were determined to be different strains based on WGS data.
- avium in patients in one pair was identified: one acquired infec- tion from the kitchen and the other from the bathroom or kitchen.
- The likelihood of patient-to-patient transmis- sion appeared minimal in these three pairs of NTM-PD patients..
- Pairs of adult patients living together and diagnosed with NTM-PD at Seoul National University Hospital satisfy- ing the following inclusion criteria were enrolled: age >.
- findings suggestive of NTM-PD on computed tomography.
- identification of NTM in ≥2 sputum cultures or in ≥1 bronchoscopic specimen.
- living in the same household prior to diagno- sis with NTM-PD.
- Swabs were taken from the inside of faucets and shower- heads in bathrooms and kitchens.
- DNA was extracted from the supernatant using a QIAamp DNA mini kit (Qiagen inc, Hilden, Germany) as previ- ously described [8].
- Identification of each isolate from the patients was performed by 16S rRNA [19] and rpoB gene [20, 21] sequencing analysis..
- High-confidence SNPs were defined as SNPs in the core regions of each NTM species.
- SNP distance and the number of different high-confidence SNPs were cal- culated for each pair of isolates.
- using only high confidence SNPs.
- The numbers of collected specimens, culture-positive, mor- phologically distinct isolates from each habitat.
- NTM-PD: Non- tuberculous mycobacterial pulmonary disease.
- WGS: Whole genome sequencing.
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