Nontuberculous mycobacteria (NTM) are environmental organisms that can cause opportunistic pulmonary disease with species diversity showing significant regional variation. In the United States, Hawai’i shows the highest rate of NTM pulmonary disease. The need for improved understanding of NTM reservoirs led us to identify NTM from patient respiratory specimens and compare NTM diversity between outdoor and indoor locations in Hawai’i. A total of 545 water biofilm samples were collected from 357 unique locations across Kaua’i (n = 51), O’ahu (n = 202), Maui (n = 159), and Hawai’i Island (n = 133) and divided into outdoor (n = 179) or indoor (n = 366) categories. rpoB sequence analysis was used to determine NTM species and predictive modeling applied to develop NTM risk maps based on geographic characteristics between environments. M. chimaera was frequently identified from respiratory and environmental samples followed by M. chelonae and M. abscessus; yet significantly less NTM were consistently recovered from outdoor compared to indoor biofilms, as exemplified by showerhead biofilm samples. While the frequency of M. chimaera recovery was comparable between outdoor and indoor showerhead biofilms, phylogenetic analyses demonstrate similar rpoB gene sequences between all showerhead and respiratory M. chimaera isolates, supporting outdoor and indoor environments as possible sources for pulmonary M. chimaera infections.

Mycobacterium chimaera causes pulmonary disease, but little is known of gradations in isolate virulence. Previously, 17 M. chimaera isolates were screened for survival in THP1 macrophages. "M. chimaera 1" was categorized as "more virulent" because it showed the greatest survival in macrophages, whereas "M. chimaera 2" was categorized as "less virulent" with reduced survival. Herein, we infected C3HeB/FeJ mice to compare the in vivo immune responses to M. chimaera 1 and 2. Unlike macrophages, significantly lower M. chimaera 1 counts were recovered from mouse lung tissue and BAL cells with less lung histopathologic changes compared to M. chimaera 2. Compared to M. chimaera 2, significantly more IL-1β, IL-6, and TNFα was produced early after M. chimaera 1 infection. LC-MS metabolomics analyses of BAL fluid revealed divergence in sphingolipid, phospholipid metabolism between M. chimaera 1 versus M. chimaera 2 mice. From pan-GWAS analyses, virulence and organizing DNA/molecular structure genes were associated with more virulent M. chimaera isolates. Vigorous lung-specific immune responses to M. chimaera 1 may influence effective bacterial control, but for a different isolate M. chimaera 2, subvert immune control. Continued studies of the gradations in virulence among the same NTM species will advance our understanding of NTM pathogenesis.