Characterization of opportunistic Pathogens in Drinking Water Supplied by Private Wells
Mapili, Kristine Irene Manzano
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Private wells are understudied potential sources of opportunistic pathogen (OP) infections. OPs, including Legionella and Mycobacterium, are of particular concern for immunocompromised individuals and are known to proliferate in drinking water systems. Much of our knowledge surrounding OP occurrence and growth in drinking water relates to municipal drinking water systems, which primarily use surface water sources and are always treated with primary and secondary disinfection in United States. However, OP occurrence and growth in private wells is not well understood and it is unclear how the knowledge developed in municipal systems will translate to private well systems with rare and infrequent exposure to chemical disinfectants. In addition, because private wells are more susceptible to microbial contamination than municipal systems, the impact of flooding on OP occurrence is of particular concern. Two private well field surveys were conducted to document the incidence of OPs in private well systems. One survey conducted in North Carolina private wells with no history of recent flooding was focused on molecular and culture-based detection of Legionella spp. and Legionella pneumophila. The other survey was a broader molecular (i.e., DNA-based) characterization of the incidence of Legionella spp., L. pneumophila, Mycobacterium spp., Mycobacterium avium (the most commonly nontuberculous mycobacteria associated with disease), and Naegleria fowleri in private wells with recent history of flooding (i.e., Hurricanes Harvey and Irma in 2017, or the Great Louisiana Floods in 2016, extending to Texas, Florida, and Louisiana). All samples in both studies were analyzed for total bacterial 16S rRNA genes, indicator bacteria (e.g., total coliform and Escherichia coli) and inorganic constituents. Information about well system characteristics were obtained through questionnaires sent to participating residents. Widespread detection of OP DNA markers were noted in the flooded well survey. Legionella spp. (detectable in 50-100% of well waters, depending on the flood event) and Mycobacterium spp. (detectable in 13.2-45.0% of well waters) were the most commonly detected among the OPs targets. At the genus level, L. pneumophila (7.9-65.5%) and M. avium (7.9-32.5%) were less commonly detected, but still highly variable. It is not possible to judge whether these OP levels were elevated as a result of the flooding because the sampling was carried out as an emergency response and background levels were not previously tested. Also of interest was whether well characteristics could predict OP levels, including well depth, well type, or treatment. However, none of these emerged as significant predictors of OP detection frequency or levels. Similarly, these OP DNA markers were not elevated in homes reporting submerged wellheads or system damage, suggesting that detection of these OPs is more dependent on the groundwater that supplies these private wells than influx of contaminated surface water. The incidence of DNA markers pertaining to N. fowleri, the "brain eating amoeba" that causes rare incidences of primary amebic meningoencephalitis (PAM), tended to be lower (5.0-12.7%) than that of other OPs targeted, but was more frequently detected in wells reporting submerged well heads, suggesting its occurrence was related to contamination from flood water. A positive association between total bacteria and occurrence of both Legionella spp. and Mycobacterium spp., was observed in private wells of all surveyed areas, contrary to observations in municipal drinking water systems with secondary disinfectant residuals. On the other hand, Legionella reportedly has an optimal growth range of 20-42 °C in municipal systems and recent surveys of municipal systems reported a strong association between Legionella spp. and temperature that was not observed for private wells in this study. We speculate that the essentially "infinite" water age and lack of disinfectant for well water, may contribute to these differences relative to municipal water supplies. The results presented in this work are likely an overestimation of OPs numbers in private wells, as molecular detection of OPs does not distinguish between live and dead cells. In addition, sample sizes were limited by laboratory throughput and budget. Identifying key variables impacting the occurrence of OPs in private wells, given that our study shows that these pathogens are relatively common, might someday help limit the risk of infections.
General Audience Abstract
Non-fecal pathogens that are capable of growth in drinking water systems and causing illness primarily in individuals with compromised immune systems are referred to as opportunistic pathogens (OPs). OPs of concern in drinking water systems in the United States include members of the Legionella genus (i.e., Legionella spp.) and Mycobacterium genus (i.e., Mycobacterium spp.), as well as Naegleria fowleri (also known as “the brain-eating amoeba”). Much of our knowledge surrounding OP occurrence and growth in drinking water relates to municipal drinking water systems. Under the right conditions, OPs have the ability to grow in municipal drinking water systems and in building plumbing systems despite the use of disinfectants. However, OP occurrence and growth in private wells is not well understood and it is unclear how the knowledge developed in municipal systems translates to private well systems that rarely utilize chemical disinfectants. In addition, because private wells are more susceptible to microbial contamination than municipal systems, the impact of flooding on OP occurrence is of particular concern. Two private well surveys were conducted to document the incidence of OP DNA markers and culturable OPs in private well systems. The first survey was conducted in North Carolina private wells with no history of recent flooding and focused on quantification of DNA markers for Legionella spp. and L. pneumophila, as well as culturable L. pneumophila. The second survey was conducted in flood-impacted private wells in vi Texas, Florida, and Louisiana following Hurricanes Harvey and Irma in 2017 and the Louisiana Floods of 2016, quantifying five DNA markers for OPs (Legionella spp., L. pneumophila, Mycobacterium spp., M. avium, and N. fowleri). All water samples in both studies were also analyzed for total bacterial numbers (i.e., total number of copies of a gene present in all bacteria), certain bacteria that indicate environmental and fecal contamination (e.g. total coliform bacteria and Escherichia coli), and inorganics. Information about well system characteristics was obtained through questionnaires sent to participating residents. While it was not possible to sample before the flooding events and determine the background detection rates, this survey found that detection of the target OPs in the flood-impacted wells was widespread, but highly variable. Both culture-based and DNAbased testing methods were used to verify the results because each approach has strengths and weaknesses. Detection of DNA markers indicates the genetic material of the organism being tested for is present and detects DNA of organisms that are both dead and alive. Thus, DNA detection may overestimate the presence live (and infectious) pathogens. Detection of culturable pathogens indicates the organism is alive, but only detects pathogens that readily grow on culture media. There may be other pathogens in the water that do not readily grow on the media. Thus culture may underestimate the occurrence of pathogens. DNA markers for Legionella spp. (detectable in 50-100% of well waters) and Mycobacterium spp. (detectable in 13.2-45.0% of well waters) were the most commonly vii detected among the targets in this study. The detection of DNA markers for L. pneumophila (7.9-65.5%) and M. avium (7.9-32.5%) was less common. There were no private well characteristics, such as well depth, well type, or treatment that emerged as significant predictors of these OP detection or levels. Similarly, these OPs were not elevated in recently flooded homes reporting submerged wellheads or system damage. Thus, detection of these OPs was found to be widespread and sporadic. Detection rates of N. fowleri, which causes rare incidences of primary amebic meningoencephalitis (PAM), DNA was lower (5.0-12.7%) than other OPs, and was also not related to private well characteristics. However, N. fowleri DNA was more frequently detected in wells with wellheads that were submerged due to flooding than in wells with unsubmerged wellheads, as were total coliform bacteria (an indicator of environmental contamination) and total bacterial numbers. This demonstrates concern that submergence of wellheads during flooding is a concern for the introduction of microbial contamination in private wells. This work also explored two trends characteristic of municipal systems that were not observed in these surveys of private wells. First, positive associations between overall bacterial numbers and DNA markers for both Legionella spp. and Mycobacterium spp. were observed in private wells in all surveyed areas. This is contrary to what has been reported for overall bacterial numbers in municipal drinking water systems with a secondary disinfectant residual. Second, Legionella has been known to have an optimal growth range of 32 to 42 °C (90 to 108 °F) in municipal systems and recent surveys of municipal systems with both a free chlorine and chloramine secondary disinfectants viii showed a strong association between Legionella spp. and temperature. These associations were not observed in private wells in this work. Continuous disinfection treatment in municipal drinking water systems may eliminate certain microbes, lowering overall levels of bacteria, while OPs may persist by resisting disinfection, resulting in no correlation between total bacteria and OPs. Private wells do not generally use continuous disinfection and represent low-nutrient environments where naturally occurring OPs can grow alongside other bacteria. The results of this study are likely an overestimation of OPs infection risk associated with private wells, as infection can only be caused by live OPs, which cannot be directly determined by measuring DNA markers. In addition, sample sizes were limited by laboratory throughput and budget. Identifying key variables that impact the occurrence of OPs in private wells is necessary to minimize the risk of associated infections linked to private wells. This work provides strong preliminary evidence that OP occurrence in private wells is relatively commonplace. Science-based options for at-risk (e.g., immunocompromised) individuals to decrease their exposure to OPs in private well water need to be developed.
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