Nontuberculous mycobacteria (NTM) are environmental organisms and opportunistic pathogens responsible for an increasingly high burden of lung disease in North America, and indeed worldwide [1
]. More than 190 NTM species have been identified to date [3
]; they have been isolated from a variety of natural environmental reservoirs, primarily soil and water. Environmental conditions related to soil properties, natural source water, and the characteristics of engineered water systems, including the biofilms that form in hospital and municipal water supplies, are believed to contribute to increased concentrations of NTM, leading to greater potential for NTM exposure [4
]. Although exposure to NTM is extremely common and the NTM disease is rare, distinct geographic variability of disease has been demonstrated in both general and high-risk populations [5
]. Hawaii, Florida and California have consistently shown high disease prevalence [8
]. These geographic differences are not explained by host-related factors, but rather are due to variation in regional environmental conditions. Specific soil and water-related factors that favor NTM growth and persistence likely increase the risk of NTM exposure within given environments. Previous epidemiologic studies [6
] demonstrate that specific environmental factors may interact to create conditions favorable for increased concentrations of NTM organisms, thereby increasing the individual exposure risk in a given environment. However, large gaps remain in our understanding of the geographic variability of NTM.
Identifying determinants of the regional ecology and environmental sources of NTM is of major public health importance [5
]. The rapidly aging U.S. population has greater risk for developing NTM disease. Explaining the increasing prevalence trends is critical, as NTM patients undergo lengthy and complex treatment regimens, and are often re-infected following initial cure. The lack of evidence-based guidance on environmental risk factors is a critical public health data gap for at-risk populations.
In our previous study [14
], we demonstrated an increased risk of NTM disease within specific watersheds in Colorado. To further explore these findings, we sought to examine why we observed higher disease risk in these areas. The aim of this study was to assess whether watershed water-quality constituents are associated with increased risk of NTM disease in Colorado. We used an ecological design with water-quality data collected or hosted by the U.S. Geological Survey, U.S. Environmental Protection Agency and National Water Quality Monitoring Council with NTM data from patients residing in the State of Colorado and treated at National Jewish Health (NJH), a leading respiratory hospital in Denver.
We found that the presence of calcium and molybdenum in the source water is associated with an increased risk of NTM disease (Table 3
, Table 4
, Table 5
and Table 6
; Models 2–5). After removing the non-significant metals from the model, we found that for every one-log unit increase in the calcium and molybdenum concentrations in the source water, a 19% and 17% increase in NTM disease risk was observed, respectively (Table 4
; Model 3). From our fitted estimates, we observe numerous high-risk watersheds in the mountainous regions to the west of the Continental Divide and along the Front Range to the east of the Continental Divide (Figure 3
; Figure 4
). Watersheds in the mountainous regions provide most of the water supply to highly populated communities in the Front Range [27
]. Molybdenum is also highly abundant in the mountainous regions of Colorado [28
The effect of molybdenum on mycobacteria has been described previously [29
]. Several molybdenum enzymes in mycobacteria exert important physiological functions. Mycobacterium tuberculosis
as well as the nontuberculous mycobacteria contain many proteins for the import and utilization of molybdenum, including the molybdate transport proteins modA, modB, and modC, and the molybdenum cofactor biosynthesis proteins moaA, moaB, moaC, moaD, and moaE. Some mycobacteria, including M. tuberculosis
, contain additional paralogs of the molybdenum cofactor biosynthesis proteins [29
]. Molybdenum has been shown to be essential for nitrate assimilation in mycobacteria [30
] and is an essential component of many bacterial enzymes involved in carbon, nitrogen, and sulfur metabolism [30
]. In M. tuberculosis
, molybdenum cofactor biosynthesis proteins have been suggested to be associated with pathogenesis [31
] and with hypoxic persistence [30
] potentially contributing to the ability to shift nitrogen respiration under the oxygen-limiting concentrations that may occur in lung granulomas. This literature suggests a physiological connection linking molybdenum and essential metabolism, potentially affecting pathogenesis and persistence of M. tuberculosis
. Although this mechanism has not been established for NTM, it offers biological plausibility because NTM and M. tuberculosis
are phylogenetically related organisms [4
Our study opens many avenues of research to investigate the influence of molybdenum on NTM growth in water sources as well as in the human host. In a recent Korean study, Oh et al. [32
] reported that trace element status is associated with mycobacterial lung disease. The authors demonstrated that patients with pulmonary NTM had higher median molybdenum concentrations in their serum (1.70 μg/L) compared with healthy controls (0.96 μg/L) and patients with pulmonary tuberculosis (0.67 μg/L). Patients and clinicians alike would benefit from knowing whether molybdenum intake from water consumption or from certain dietary profiles (e.g., vitamin supplementation containing molybdenum) increases the risk of infection and/or progression of disease.
Molybdenum is mainly used as an alloying agent in the production of steel because of its strength and ability to withstand high temperatures. Small quantities of molybdenum are essential to human, animal and plant life, and it is present in trace quantities in rocks, soil and water, often at concentrations less than 10 µg/L [33
]. The environmental concentrations of molybdenum can vary widely, and in places where molybdenum is processed, the concentrations in soil and water may increase considerably [34
]. Molybdenum has “relatively high geochemical mobility—a tendency to enter into solution in water under normal Earth-surface conditions” [34
]; we hypothesize that perhaps even small amounts of water-soluble molybdenum may act as a metabolic source for NTM in the water supply. Soil moisture is known to influence molybdenum availability: poorly drained wet soils (for example, peat marshes, swampy organic rich soils) tend to accumulate molybdenum to high levels [36
]. Likewise, Falkinham and colleagues have repeatedly shown that peat rich soils and brackish marshes are rich in NTM [4
]. In addition, molybdenum can form complexes with organic matter, particularly humic and fulvic acids [39
]. Falkinham and colleagues have also reported that humic and fulvic acids support high numbers of Mycobacterium avium
complex (MAC) species [4
]. We found that the median value of molybdenum across Colorado watersheds was 4.3 µg/L (Table 1
), but reached 325 µg/L at one specific watershed.
Many studies have examined mycobacterial distributions and abundance in different geographic areas. These studies range from examining the presence of NTM in premise plumbing [6
], in the water distribution systems [44
], in the water treatment facilities [45
] and in the watershed untreated source water [46
] (Figure 5
Studies have demonstrated that NTM exposure and infection occurs in the home (Figure 5
], with household water as a source of exposure. Lande et al. [41
], for example, showed genotypic matches between M. avium
respiratory isolates and isolates from household plumbing. Studies have shown the proliferation of NTM in water distribution systems upstream from premise plumbing [44
] (Figure 5
B). However, the entry point for these organisms into the water distribution system and premise plumbing remains unknown. Farther upstream, NTM have been found in water treatment facilities (Figure 5
C). King et al. [45
] conducted a survey to obtain information on mycobacteria (as well as other microbial pathogens) in source and treated drinking water collected from drinking water treatment plants (DWTPs) across the United States (Figure 5
C,D). M. avium
and Mycobacterium intracellulare
were detected in 6 out of 24 source water samples and both samples were detected simultaneously at 4 DWTPs. King et al. also identified 10 out of 24 DWTPs that had no mycobacteria detected in source or treated water. The literature indicates that the high-risk and low-risk regions for mycobacterial exposure likely correspond to high and low risk areas for disease.
Although we did not find literature that explain the association of calcium in source water on NTM disease risk, we speculate that for this Colorado dataset the association may be explained in part by the correlation of calcium with molybdenum because of environmental factors, rather than any meaningful link between calcium and NTM bacteria or NTM disease prevalence. Watersheds with greater disease risk and dissolved molybdenum concentrations are more prevalent on the eastern plains of Colorado (Figure 4
). Compared to much of Colorado, waters on the eastern plains have generally greater concentrations of major constituents, like calcium, because of evapotranspiration in a warmer and drier climate, and more prevalent sedimentary rocks that weather to release calcium [50
]. An association of such rocks with molybdenum release remains to be explored and warrants further research.
Additional research is required to confirm the causal pathway between molybdenum, NTM abundance, and disease prevalence. If molybdenum is a metabolic factor for NTM in the environment, it is plausible that the mycobacteria also utilize this trace metal to survive in the host, a possibility that may explain reports of higher blood-serum molybdenum concentrations in NTM patients [32
]. Identification of these factors is critical to develop prevention strategies for minimizing exposure and infection in high-risk regions.