ABSTRACT
Potable water can be a source of transmission for legionellosis and nontuberculous mycobacterium (NTM) infections and diseases. Legionellosis is caused largely by Legionella pneumophila, specifically serogroup 1 (Sg1). Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus are three leading species associated with pulmonary NTM disease. The estimated rates of these diseases are increasing in the United States, and the cost of treatment is high. Therefore, a national assessment of water disinfection efficacy for these pathogens was needed. The disinfectant type and total chlorine residual (TClR) were investigated to understand their influence on the detection and concentrations of the five pathogens in potable water. Samples (n = 358) were collected from point-of-use taps (cold or hot) from locations across the United States served by public water utilities that disinfected with chlorine or chloramine. The bacteria were detected and quantified using specific primer and probe quantitative-PCR (qPCR) methods. The total chlorine residual was measured spectrophotometrically. Chlorine was the more potent disinfectant for controlling the three mycobacterial species. Chloramine was effective at controlling L. pneumophila and Sg1. Plotting the TClR associated with positive microbial detection showed that an upward TClR adjustment could reduce the bacterial count in chlorinated water but was not as effective for chloramine. Each species of bacteria responded differently to the disinfection type, concentration, and temperature. There was no unifying condition among the water characteristics studied that achieved microbial control for all. This information will help guide disinfectant decisions aimed at reducing occurrences of these pathogens at consumer taps and as related to the disinfectant type and TClR.
IMPORTANCE The primary purpose of tap water disinfection is to control the presence of microbes. This study evaluated the role of disinfectant choice on the presence at the tap of L. pneumophila, its Sg1 serogroup, and three species of mycobacteria in tap water samples collected at points of human exposure at locations across the United States. The study demonstrates that microbial survival varies based on the microbial species, disinfectant, and TClR.
INTRODUCTION
Legionella and Mycobacterium are two genera of waterborne environmental bacteria that affect human health by causing the respiratory diseases legionellosis (Legionnaires’ disease and Pontiac fever) (1) and nontuberculous mycobacterium (NTM) infections and diseases (2), respectively. Potable water is often suspected or identified as the cause of individual cases or a cluster of cases (an outbreak). Outbreaks for both genera may occur at health care facilities (3, 4). Transmission of both diseases requires inhalation of the bacteria contained in aerosols or, to a lesser degree, aspiration.
The reported incidence rate of legionellosis in 2015 was 1.6 cases/100,000 persons in the United States (5) and 1.4 cases/100,000 persons in Europe (22 countries) (6). The prevalence rate of NTM is not available because mycobacterial diseases, other than tuberculosis and leprosy, are not required to be reported to health authorities on a national level in most countries, including the United States. However, North America has a greater prevalence of NTM disease (5.5 to 9.8 cases/100,000 persons) (7) than Europe and other countries (0.6 to 3.1 cases/100,000 persons) (8–10). The reported incidence rates of the respiratory diseases associated with both microbial genera are increasing in North America and Europe (11–14).
Despite chemical disinfection of source waters by utilities, both Legionella spp. and Mycobacterium spp. are recovered from potable-water samples (15, 16). Legionella spp. are susceptible to both chlorine (Cl) (17) and monochloramine (CLM), hereafter referred to as chloramine (18–20). Mycobacterium spp. are more resistant to both disinfectants (21–23). Disinfectant effectiveness may also be influenced by physiochemical parameters, such as pH and temperature, and by the water’s nutrient composition (organic carbon and nitrogen) (17, 21). This survey was undertaken to examine the differences in the use of chlorine and chloramine as chemical disinfectants and to correlate their residual concentrations with the concentrations of the microbes present in samples collected at consumer taps.
Legionella pneumophila and L. pneumophila serogroup 1 (Sg1) (causing legionellosis) and Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus (causing pulmonary NTM disease) were quantified in water samples taken at locations across the United States. The detection frequencies and concentrations of the pathogens in samples collected from both the cold- and hot-water lines were compared. The goal was to examine the effectiveness of chlorine and chloramine in controlling microbial survival.
RESULTS
Pathogen detection frequency.Table 1 compares the detection frequency of each of the pathogens in tap water samples from systems that used chlorine to those in samples from systems that used chloramine for disinfection. When all chlorine- and chloramine-treated water samples were considered, L. pneumophila and L. pneumophila Sg1 were detected at similar rates (26% [CL] versus 22% [CLM] for L. pneumophila and 9% [CL] versus 5% [CLM] for L. pneumophila Sg1). M. avium and M. abscessus were detected (22% and 17%) significantly more frequently at locations where chloramine was used as the disinfectant (P = 0.02 and 0.03). Detections of the three Mycobacterium spp. ranged from 9 to 22%. However, M. avium and M. abscessus were detected significantly more frequently in chloramine-treated water samples than in chlorine-treated samples (Table 1). All the pathogens showed no significant difference in detection frequency in hot-water or cold-water tap samples whether from chlorine- or chloramine-treated water sources.
Numbers and percentages of samples positive for each bacterial species
Pathogen concentrations.Table 2 shows the median numbers of cell equivalents (CE) per liter for all samples. Samples from the hot-water and cold-water taps were analyzed separately. There were significantly fewer L. pneumophila CE per liter detected in samples from chloramine-treated water than in chlorine-treated samples (P < 0.001). This was also true for samples collected from cold water compared to those collected from hot water. The median concentrations for L. pneumophila Sg1 and for the three Mycobacterium species were not significantly different based on the two disinfectant types. Although a large difference was observed in the median concentration of L. pneumophila Sg1 for chlorine compared to chloramine (Table 2), the difference lacks statistical significance due to the small number of positive samples. Among the mycobacterium species, M. intracellulare concentrations were significantly greater in chloramine-treated systems than in chlorine-treated systems in cold-water samples.
Microbial concentration comparison to the disinfectant used and cold- and hot-water lines
Figure 1 shows the concentrations (CE per liter) for the microbial targets detected based on the disinfectant used and for the cold-water lines compared to the hot-water lines (see supplemental material at https://catalog.data.gov/dataset/Disinfetant-Impact).
Concentrations of target by chemical disinfectant and by cold- or hot-water line. (A) L. pneumophila. (B) L. pneumophila Sg1. (C) M. avium. (D) M. intracellulare. (E) M. abscessus.
TClR.Table 3 shows the total chlorine residual (TClR) median and average concentrations for chlorine and chloramine and for hot- and cold-water line samples. Chlorinated-water sample TClR concentrations were lower than the concentrations detected in chloramine-treated water samples. Measurable TClR was more likely to be present in cold-water samples than in hot-water samples (94 to 96% versus 71 to 88%, respectively).
Summary of the results of total chlorine residual measurements in chlorine- and chloramine-treated hot- and cold-water samples
Table 4 presents the impact that the TClR concentration had on the detection frequency for the measured microbes. A positive (P)/negative (N) sample ratio was created for each TClR concentration category. A positive sample was any sample that was positive for one or more of the microbes measured. A negative sample was any sample that had no pathogen detected. In the chlorine-treated water, the detection frequency decreased as the TClR increased. This was not the case for chloramine-treated samples. Although some level of TClR was detected in most chloramine-treated samples, the P/N ratio remained about 1:1 regardless of the TClR concentration. However, in one TClR category (1 to 1.5 mg/liter), the P/N ratio was 4:1 in chloramine-treated water samples and was statistically significantly (P = 0.005) different from the P/N ratio (1:3) for chlorine-treated water samples in the same TClR category. However, when the P/N ratios for samples from cold- and hot-water taps were compared, there was no statistically significant difference in pathogen occurrence by disinfectant type (Table 5).
Changes in pathogen occurrence resulting from differences in total chlorine residual in chlorine- or chloramine-treated water at hot- and cold-water tapsa
Changes in pathogen occurrence resulting from differences between water taken from cold- and hot-water taps for each disinfectant typea
Figure 2 graphically plots the P/N ratio as a function of the TClR in water samples from locations where potable water came from utilities disinfecting with chlorine or chloramine. For the chlorine systems, there was a significant decrease in pathogen detection at a TClR level of up to 1.5 mg/liter but no further significant change at higher TClR levels (Fig. 2A). This was observed in both cold- and hot-water line samples. For chloramine systems, the response in the cold-water systems differed from the response in the hot-water samples (Fig. 2B). In the cold water, the P/N ratio increased as the TClR increased to levels of 1.0 to 1.5 mg/liter and decreased at concentrations greater than 1.5 mg/liter. In the hot water, the P/N ratio remained relatively constant.
Ratios of the number of positive (pathogen) samples to the number of negative samples by TClR category. (A) Chlorine. (B) Chloramine.
DISCUSSION
Neither chlorine nor chloramine eliminated pathogenic Legionella or Mycobacterium organisms in the water samples tested. Although L. pneumophila and L. pneumophila Sg1 were detected at about the same frequency in systems that disinfected with chloramine or chlorine (Table 1), chloramine significantly reduced the median concentration of L. pneumophila compared to chlorine-treated samples for both the cold- and hot-water samples plus all samples combined (Table 2). On the other hand, M. avium and M. abscessus were detected significantly more frequently in water samples from systems that disinfected with chloramine than from those using chlorine (Table 1), but the median concentrations were not significantly different for chloramine-treated water samples versus chlorine-treated water samples (Table 2). The median concentrations of L. pneumophila Sg1 were significantly lower only in cold-water samples from utilities that used chloramine compared to those that used chlorine (Table 2). Although a large difference in the median concentration of L. pneumophila Sg1 was observed between chlorine- and chloramine-treated samples, as shown in Table 2, the difference lacks statistical significance due to the low number of positive samples. The median concentrations of M. intracellulare in cold-water samples were significantly higher for chloramine-treated samples than for chlorine-treated samples (Table 2). These results appear to contradict the findings from controlled laboratory studies, which suggest both chlorine and chloramine are effective in eliminating the pathogens. However, laboratory studies can be confounded by the presence of either amoeba-encysted microbes or the nutrients in biofilm that are typically present in water from potable systems and are known to influence microbial survival (24, 25). In our study, disinfectant effectiveness appeared to vary widely across the United States. The varying degrees of microbial susceptibility to the disinfectants may be related to the characteristics of the different source waters (reservoirs, lakes, rivers, etc.), which are highly diverse in their chemical compositions (e.g., concentrations of organic carbon and nitrogen) and physical properties (e.g., pH and temperature). As a disinfectant is oxidized or metabolized, its antimicrobial potency can change. Also, higher concentrations of organic matter may consume some of the oxidizing potential of the disinfectant. These factors affect the TClR and may also impact the levels of carbon-containing disinfection by-products (DBPs) (e.g., trihalomethane and haloacetic acid), illustrating the need to balance DBPs and the total chlorine residual.
In the early 1900s, chemical disinfection practices were initiated by water utilities to reduce outbreaks of waterborne diseases (26). In 1989, the U.S. Environmental Protection Agency (EPA) enacted the first Surface Water Treatment Rule (SWTR) to require maintenance of a “measurable” level of disinfectant (0.2 mg/liter) within the distribution system (27). However, there is no federal guidance on maintaining a specific TClR (26). TClR is officially defined as anything from “detectable” to 1.5 mg/liter by some states (28). In this study, 74% of chlorinated and 89% of chloramine-treated cold-water samples had TClR concentrations exceeding 0.2 mg/liter. This value was more effective at pathogen control in chlorine-treated than in chloramine-treated water (<0.2 versus >0.2 mg/liter; Cl, 70% versus 34% [P = 0.085]; CLM, 25% versus 52% [P = 0.585]).
In the United States, chlorine is the most widely used disinfectant. It has been shown that chloramine treatment requires higher TClR concentrations and longer contact times to obtain the same level of microbial control as with chlorine. In general, utilities that disinfected with chloramine controlled the pathogens less well than those that used chlorine (Table 4 and Fig. 2). Total chlorine residual criteria are applied widely with little consideration given to the disinfectant responsible for the residual. Based on the data collected, adjusting the TClR, especially the free-chlorine component of the TClR, has the greatest potential benefit in controlling Legionella and Mycobacterium pathogens.
However, higher levels of either chlorine or chloramine can cause other water quality issues, such as objectionable taste and chlorine odor or skin and eye irritation (29). Chloramine treatment may promote the growth of nitrifying bacteria. Thus, the choice of a disinfectant is complex, with a need to balance multiple factors as they relate to the microbial species, water chemistry, DBP levels, and complexity of the distribution system. As a result, many utilities disinfecting with chloramine periodically perform a “chlorine burn” or switch to free chlorine for only a limited period. This temporary change from chloramine to chlorine may provide an opportunity for L. pneumophila to proliferate (30). Therefore, finding the right disinfectant for a specific location is critical.
A strength of this study is that samples were taken at point-of-use taps, where human exposures happen (31). A benefit of quantitative PCR (qPCR) is the fact that it can detect microbes in other physiological states, including amoeba-encysted and viable but nonculturable cells. These states are not detected by culture. Therefore, qPCR can quantify concentration levels below those quantifiable by culture. A weakness of qPCR is that it can detect DNA from dead cells. Despite this limitation, qPCR is considered a reasonable approach for quantification of potential risk.
When culturing and qPCR are compared, they provide comparable results. For instance, L. pneumophila concentrations in the hot-water samples were higher than in the cold-water samples (32). The qPCR-based observations in this study are consistent with the culture-based linear response to the chlorine concentration. Further, the culture-based measures of chloramine’s effectiveness in reducing L. pneumophila concentrations (18, 20, 33) and lack of effectiveness in controlling Mycobacterium spp. (22, 23, 34, 35) are consistent with our qPCR-based findings. Therefore, the use of qPCR for the identification and quantification of these pathogens is not a major methodological limitation.
This study demonstrates factors related to the complexity of disinfectant selection. As a disinfectant is oxidized or metabolized, its antimicrobial potency changes. This study shows that disinfectant selection needs to be considered and tested in a site-specific manner. In the case of chlorine, TClRs at levels of 0.1 to >1.5 mg/liter were equivalently effective at reducing the survival of both L. pneumophila and Mycobacterium species (Fig. 2). In the case of chloramines, the data show that each microbial species responded differently. Chloramine reduced L. pneumophila survival but enhanced the detection frequency of the Mycobacterium species, independently of the TClR. Although exact temperature measurements were not taken, Mycobacterium survival in cold water exceeded its detection frequency in hot water at moderate TClR concentrations.
MATERIALS AND METHODS
Study design.Sampling occurred between 2011 and 2017. Water samples (358) came from 46 states and territories across the United States. Two hundred and ten samples came from taps whose water was chlorinated, and 148 water samples were from taps where the water was chloraminated (Fig. 3). The sampling locations had no known association with outbreaks of legionellosis or pulmonary NTM disease and were not statistically selected. Volunteers collected the water samples. Water quality reports and/or consumer confidence reports identified the disinfectant used by the public water utility at the sample location. See “Chlorine residual” below for specifics.
Number of samples by variable.
Water samples.Three 1-liter high-density polypropylene (HDPP) bottles were filled with water from each tap used to collect the cold- and hot-water samples. The water was collected after a 15-s flush time. The samples were packed with ice packs on the day of sample collection and immediately sent by next-day air to an EPA research laboratory (Cincinnati, OH).
Water samples for qPCR.Upon sample arrival, 3 liters of the water was vacuum filtered through a sterilized Whatman Nuclepore track-etched membrane (47 mm; 0.4-μm polycarbonate membrane; Whatman Inc., Piscataway, NJ). After filtration, the polycarbonate membrane filter was inserted into a sterile 2-ml O-ring screw cap microcentrifuge tube containing 0.30 ± 0.05 g of 0.1-mm sterile glass beads (BioSpec Products, Bartlesville, OK). The study generated 358 samples, not including method blanks, extraction blanks, and positive and negative controls. The bead tubes containing membrane filters were stored at −80°C until they were needed.
DNA extraction.Details of DNA extraction from membrane filters were published previously (36). Briefly, each polycarbonate membrane was beaten with minibeads in a bead beater (BioSpec Products, Bartlesville, OK) with 500 μl of tissue and cell lysis solution (Lucigen Corporation, Middleton, WI). The lysate was transferred, and 2 μl of proteinase K (50 μg/μl; Lucigen Corporation, Middleton, WI) was added, followed by incubation at 65°C in a water bath for 15 min. Next, 2 μl of RNase A (5 μg/μl; Lucigen Corporation, Middleton, WI) was added to the mixture and incubated at 37°C for 30 min. Subsequently, 350 μl of MPC protein precipitation reagent (Lucigen Corporation, Middleton, WI) was added to precipitate the cellular proteins. The resulting supernatant was transferred to a microcentrifuge tube with an equal volume of ice-cold isopropanol (approximately −4°C). The samples were inverted manually up to 40 times and centrifuged at 10,000 × g for 10 min. The isopropanol was poured off, and the resulting DNA pellet was washed with 500 μl of ice-cold (approximately −4°C) 70% ethanol. Samples were centrifuged, and the ethanol was removed. The DNA pellets were resuspended in 150 μl of nuclease-free sterile water and stored at −80°C until they were analyzed.
Preparation of qPCR standard/positive control.The previously published instructions for the preparation of the DNA standards for the qPCR method (16, 36) are described below.
Assays and conditions for qPCR.Primer-probe sets were used to detect and quantify L. pneumophila (16), L. pneumophila Sg1 (37), M. avium (38), M. intracellulare, and M. abscessus (39) in water. All DNA extracts were analyzed using the L. pneumophila 16S, M. avium, M. intracellulare, and M. abscessus primer-probe sets (Table 6). Any extract that was positive for L. pneumophila was also analyzed for the presence of L. pneumophila Sg1 using the L. pneumophila Sg1 primer-probe set. The details of all five primer-probe sets and qPCR conditions were previously published (16, 38). The following qPCR conditions and instrument were used for the L. pneumophila and L. pneumophila Sg1 assays. The qPCR total reaction volume was 25 μl and consisted of 17 μl of TaqMan Universal PCR Master Mix plus 2 μl of a mixture of forward and reverse primers (500 nM), a 100 nM Integrated DNA Technologies (IDT) probe, 1 μl of 0.1% bovine serum albumin (BSA), and 5 μl of purified DNA. Five microliters of DNA extract was analyzed per qPCR, equal to 100 ml of the original sample volume.
Sequences of primers and probes used for assays
Reactions were performed in triplicate for the L. pneumophila assay and in duplicate for the L. pneumophila Sg1 assay using a Roche LightCycler 480 II real-time PCR system (Roche Applied Science, Indianapolis, IN). The LightCycler setting included monocolor hydrolysis detection with a preincubation step of 10 min at 95°C; an amplification step of 40 cycles of 10 s at 95°C, 30 s at 60°C, and 1 s at 72°C; and a cooling step of 30 s at 40°C.
For the M. avium, M. intracellulare, and M. abscessus assays, amplification was performed using 10 μl DNA extract, 12.5 μl TaqMan Environmental Master Mix 2.0 (Applied Biosystems, Foster City, CA), 2.5 μl exogenous internal positive control (IPC) mix, 0.5 μl exogenous IPC DNA, 1.0 μl 0.5-mg/ml bovine serum albumin, 900 nM each primer, and 200 nM probe in a total volume of 35 μl. All reactions were carried out in an ABI model 7900 HT sequence detection system (Applied Biosystems, Foster City, CA) in triplicate. The thermocycler conditions consisted of 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. The data were analyzed with the quantification cycle (Cq) threshold set at 0.01.
Interpretation of qPCR.A sample was considered positive for a specific microorganism if two or more of the triplicate reactions had a Cq value of <39. For samples tested with the L. pneumophila Sg1 assay, both replicates were required to have Cq values of <39 to be considered positive.
Controls.Controls were included on each plate to ensure the integrity of the method and confidence in the results. Genomic DNA extracted from L. pneumophila Sg1 ATCC 33152, M. avium ATCC 76102, M. intracellulare ATCC 13950, and M. abscessus ATCC 19977 (American Type Culture Collection, Manassas, VA) was used as a positive control for each respective assay. A serial dilution of seven concentrations ranging from 106 to a theoretical 1 genomic copy was made from the DNA. Negative-control measures included three nontemplate controls (NTC), where sterile water was used in place of the DNA extract (template).
Method blanks were established at the time of water filtration. One hundred milliliters of sterile molecular-grade water (5 Prime, Gaithersburg, MD) was vacuum filtered as described above for every 10 samples filtered. If a method blank was positive, the set of samples that corresponded to it was considered compromised, and the data were discarded. Inhibition of the qPCR was monitored using external controls for the L. pneumophila and L. pneumophila Sg1 assays and an internal control for the M. avium assay. The external inhibition control was prepared as follows: all unknown samples were spiked with 1 μl of an exogenous control of 10,000 target gene copies extracted from L. pneumophila ATCC 33152. A reaction was considered inhibited if the observed Cq value of either the external or internal control drifted ≥1.5 Cq units from the standard Cq value.
Chlorine residual.The TClR and monochloramine concentrations were colorimetrically measured using Hach’s (Loveland, CO) total chlorine and Monochlor F reagents. These analyses were performed immediately upon sample arrival. Residual testing was not performed at the sampling location because a spectrophotometer was needed to read the results. Ten milliliters of water was used for each test. Manufacturers’ instructions were followed, and a Hach’s (Loveland, CO) 3600 spectrophotometer was used to measure the colorimetric results.
Statistical analysis.The Cq values were initially transformed into genomic target numbers using a standard curve based on a known number of cells. The average of the genomic targets per replicate was calculated for each sample that had a Cq of <39. The detection frequencies of each pathogen in chlorine- and chloramine-treated samples and from water taken from the cold- and hot-water lines were compared using either a Fisher exact test or chi-square analysis (Sigma Plot 13.0; Systat, San Jose, CA). For studies comparing disinfectants and cold- and hot-water concentrations, the Mann-Whitney U test was used for statistical comparisons. Chi-square analysis was used to compare the five TClR concentration levels: <0.1, 0.1 to 0.5, 0.5 to 1.0, 1.0 to 1.5, and >1.5 mg/liter.
ACKNOWLEDGMENTS
M.J.D. thanks Dawn King for performing the DNA extractions from 2011 to 2013.
The U.S. Environmental Protection Agency through its Office of Research and Development funded and managed the research described here. It has been subjected to the Agency’s administrative review and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
FOOTNOTES
- Received 26 August 2019.
- Accepted 3 October 2019.
- Accepted manuscript posted online 11 October 2019.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
