Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses

Abstract

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Fig. 1

Overall species diversity observed within Colorado environmental samples from 44 recovered NTM isolates

Fig. 2

NTM species diversity based on rpoB sequencing identification and stratified by a Type of sample collected. “n” identifies the number of each particular sample that was NTM positive and the diversity of species found in that sample type. For example, there were 12 NTM positive ice machines harboring seven different species (seven different colored bars) in varying proportions. b NTM species diversity modeled by risk area for Colorado NTM LD. “n” identifies the number of different NTM positive locations sampled per risk region

Fig. 3

Attributes of the 11 Colorado M. chelonae isolates isolated from the same number of unique sites. a The proportion of environmental sample types from which M. chelonae was microbiologically recovered. b Colorado cities from which the most M. chelonae was recovered; M = moderate risk area, H = high risk area, L = low risk area

Fig. 4

Phylogenomic tree of MinION whole genome sequenced M. chelonae isolates from Colorado and a Hawai'i environmental sample

Fig. 5

Similarity matrix of identified prophages. Higher ANI (lighter coloration) indicates more sequence similarity