Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing.

BACKGROUND: Nontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques provide new opportunities to sequence and construct complete bacterial genomes faster and at a lower cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to its slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA with no additional clean-up steps. RESULTS: Our DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. We tested our optimized method on 38 clinical isolates from the M. avium and M. abscessus complexes, which yielded optimal quality and quantity measurements for Oxford Nanopore Technologies sequencing. We also present the efficient completion of circularized M. avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid. CONCLUSIONS: Our optimized extraction protocol and assembly pipeline was both sufficient and efficient for genome closure. We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.

Household proximity to water and nontuberculous mycobacteria in children with cystic fibrosis.

BACKGROUND: Nontuberculous mycobacteria (NTM) have a particular affinity for patients with cystic fibrosis (CF). Recent studies suggest a possible relationship between acquiring NTM and the level of environmental water in a given area. We sought to determine if there is an association between household proximity to water and NTM in children with CF. MATERIALS AND METHODS: An IRB-approved retrospective chart review was completed on 150 children with CF in Florida. Inclusion criteria required regular follow-up, at least two acid-fast bacilli cultures, and a consistent home address over a 3-year period. The distance from each patient's home to the nearest body of water was measured using ArcMap®, a Geographic Information System, and the mean distance to water for NTM-positive and NTM-negative groups were compared. A stepwise backwards logistic regression was used to evaluate for predictors of NTM-positivity. RESULTS: Of the 150 CF patients, 65 met inclusion criteria and 21 (32.3%) tested positive for NTM. Comparison of the mean distance to water for NTM-positive versus NTM-negative groups revealed a cutoff of 500 meters. On the logistic regression, CF patients who lived within 500 meters of water were 9.4 times more likely to acquire NTM (P = 0.013). Other significant predictors included a history of Aspergillus fumigatus (OR 7.9, P = 0.011) and recent history of Pseudomonas aeruginosa (OR 2.5, P = 0.007). CONCLUSIONS: In the regions studied, children with CF who live closer to water are more likely to acquire nontuberculous mycobacteria. Future studies in other geographic areas are needed to determine if these results are generalizable. Pediatr Pulmonol. 2017;52:324-330. © 2016 Wiley Periodicals, Inc.