Article pubs.acs.org/est
Widespread Molecular Detection of Legionella pneumophila Serogroup 1 in Cold Water Taps across the United States Maura J. Donohue,*,† Katharine O’Connell,‡ Stephen J. Vesper,† Jatin H. Mistry,§ Dawn King,† Mitch Kostich,† and Stacy Pfaller† †
Office of Research and Development, National Exposure Research Laboratory, United States Environmental Protection Agency, 26 West Martin Luther King Drive, Mail Stop 593, Cincinnati, Ohio 45268, United States ‡ ORISE Fellow, Office of Research and Development, National Exposure Research Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio 45268, United States § Region 6, Drinking Water Section, United States Environmental Protection Agency, Dallas, Texas 75202, United States S Supporting Information *
ABSTRACT: In the United States, 6,868 cases of legionellosis were reported to the Center for Disease Control and Prevention in 2009−2010. Of these reports, it is estimated that 84% are caused by the microorganism Legionella pneumophila Serogroup (Sg) 1. Legionella spp. have been isolated and recovered from a variety of natural freshwater environments. Human exposure to L. pneumophila Sg1 may occur from aerosolization and subsequent inhalation of household and facility water. In this study, two primer/probe sets (one able to detect L. pneumophila and the other L. pneumophila Sg1) were determined to be highly sensitive and selective for their respective targets. Over 272 water samples, collected in 2009 and 2010 from 68 public and private water taps across the United States, were analyzed using the two qPCR assays to evaluate the incidence of L. pneumophila Sg1. Nearly half of the taps showed the presence of L. pneumophila Sg1 in one sampling event, and 16% of taps were positive in more than one sampling event. This study is the first United States survey to document the occurrence and colonization of L. pneumophila Sg1 in cold water delivered from point of use taps.
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INTRODUCTION Legionella pneumophila is an environmental microorganism capable of causing a range of adverse health effects from severe Legionnaires’ disease to moderate Pontiac fever influenza-like symptoms in humans. In 2009−2010, CDC’s National Notifiable Disease Surveillance System (NNDSS) reported 6,868 cases of legionellosis.1,2 Only 2.6% (179/6,868) of these cases were outbreak events associated with drinking water, other nonrecreational waters, or recreational waters.3,4 The remaining 97.4% cases of legionellosis were characterized as sporadic (e.g., not associated with an outbreak). According to the CDC’s outbreak reports, exposure to Legionella is through potable water when other nonrecreational uses such as ornamental fountains and cooling towers are included3,4 with drinking water. Independent of exposure route, legionellosis is primarily acquired from aerosolized water droplets contaminated with Legionella microorganisms. Water contamination by Legionella is currently monitored by many countries utilizing conventional culture methods based on International Organization for Standardization (ISO) 11731,5 The Netherlands Normalizatie-Instituute (NEN) 6265,6 or The Association Française de Normalization (AFNOR) NF T90-4317,8 methods. However, as is the case with all methods, there are limitations when culturing This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society
Legionella. Optimal cultivation requires amino acid supplementation, attention to pH sensitivity, and prolonged incubation periods (up to ten days). With a generation time of 4 to 6 h, overgrowth of cultures with other microorganisms is likely to occur.9,10 Another factor limiting the culture-based detection of Legionella is the physiological state of the cells. For instance, viable but nonculturable (VBNC) Legionella cells and cells present within amoeba will not be detected by conventional culture methods.11,12 The use of qPCR for the purpose of surveillance has some merit because qPCR detects and amplifies a specific gene target known to be exclusive to a specific genus/species/serogroup. The qPCR technique, with the use of a standard curve, is far more effective and efficient at quantifying the presence of a specific microorganism than the traditional culture approach. To date, several qPCR assays for Legionella have been developed: a genus-specific assay (targeting the 16S rRNA gene from Legionella spp.),13 a species-specific assay (targeting the mip gene, coding the macrophage infectivity-potentiator of Received: Revised: Accepted: Published: 3145
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L. pneumophila),13−15 and a species/serogroup-specific assay(targeting the LPS-gene cluster from L. pneumophila Sg1).16 Assays have also been developed targeting the 5S rRNA gene,17−20 the 23S rRNA gene,21 the rpoB gene,22 and the dotA gene.23 QPCR allows for rapid detection with high specificity and sensitivity. However, because DNA can persist in the environment for hours after cell death, qPCR cannot distinguish between viable and nonviable cells.24,25 This limitation can result in an overestimation of the potential exposure and associated risk due to false-positive results. Despite the known drawbacks, qPCR can provide information on the maximum potential exposure levels detected in potable water. It is also useful in a scenario where low levels of L. pneumophila Sg1 are constantly occurring but are undetectable by culture. In this study, two qPCR assays (Lp16S and LpLPS) were evaluated for the detection of L. pneumophila Sg1 in potable water. These assays were employed to investigate occurrence and colonization of L. pneumophila Sg1 in household and facility cold water taps across the U.S.; thus, providing the exposure data (frequency and concentration) necessary to estimate potential risk associated with this microorganism.
In 2009, water was collected from each tap three times during the year. In 2010, water was collected once from the same sites. At all taps, the water was collected in three, one liter high density polypropylene (HDPP) bottles, fifteen seconds after the water started flowing; sodium thiosulfate was not present in the sample collection bottle. The fifteen second flush was performed to ensure that the water collected came from the cold line behind the hot and cold interface. The water was shipped next day delivery with ice packs, and samples were vacuum filtered within 48 h of its collection. Water quality reports from the water distribution authority were obtained for the locations (n = 38) represented by the samples. Samples and DNA Extraction Used for qPCR Analysis. Two hundred and seventy-two DNA extracts were generated from the collected samples. The extracts were the result of vacuum filtering 3 L of water through a sterilized Nucleopore Track-Etch, 47 mm, 0.4 μm polycarbonate membrane (Whatman Inc., Piscataway, NJ). The filters were stored at −80 °C in sterile 2.0 mL O-ring screw cap microcentrifuge tubes containing 0.30 ± 0.05 g 0.1 mm of glass beads (BioSpec Products, Bartlesville, OK) until extraction. The DNA was extracted from each sample using a modified WaterMaster DNA extraction protocol (Epicenter Biotechnologies, Philadelphia, PA). Details of this extraction have been published previously.26 Briefly, each polycarbonate membrane was bead-beaten in a bead-beater (BioSpec Products, Bartlesville, OK) for 3 min on the “homogeneity” setting with 500 μL of Tissue and Cell Lysis Solution (Epicenter Biotechnologies, Philadelphia, PA). The lysate was transferred to 2 mL microcentrifuge tubes, and 2 μL of Proteinase K (50 μg/μL) (Epicenter Biotechnologies, Philadelphia, PA) was added and incubated at 65 °C in a water bath for 15 min. Next, 2 μL of RNase A (5 μg/μL) (Epicenter Biotechnologies, Philadelphia, PA) was added followed by incubation in a 37 °C water bath for 30 min. Samples were placed on ice for 5 min, and 350 μL of MPC Protein Precipitation Reagent (Epicenter Biotechnologies, Philadelphia, PA) was added. The supernatant was transferred to a sterile microcentrifuge tube, and an equal volume of ice cold (∼−4 °C) isopropyl alcohol was added. The samples were inverted manually up to 40 times and then centrifuged at 10,000g for 10 min. The isopropyl alcohol was poured off, and the resulting DNA pellet was washed with 500 μL of ice cold (∼ −4 °C) 70% ethanol. Samples were centrifuged, and the ethanol was removed. The pellets were resuspended in 150 μL of nuclease-free sterile water and stored at −80 °C until analyzed. Bacterial Culture and Preparation of Standard. L. pneumophila Sg1 ATCC 33152 (ATCC, Manassas, VA) and other strains (Table S1) were cultured on Buffered Charcoal Yeast Extract (BCYE) agar base containing 10% L-cysteine (BD, Franklin Lakes, NJ) at 35 °C under microaerobic conditions (Mitsubishi Gas Chemical America, New York, NY). After 48 h, a 10 μL loopful of cells was harvested from BCYE agar plates, and the DNA was extracted as described above. The extracted DNA was quantified on a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). For the specificity tests, DNA was diluted to 1 ng/μL and stored at −80 °C until analyzed by both assays. A standard curve was prepared by using L. pneumophila Sg1 ATCC 33152 by calculating 106 target gene copies per qPCR reaction from the 4.3 fg genomic mass27 of L. pneumophila. Next, a (1:10) serial dilution was performed down to a “theoretical” one target gene copy per qPCR reaction. In each
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MATERIALS AND METHODS Sampling Sites. Forty geographically dispersed sites (32 buildings and 8 houses) in 25 states, 1 territory, and 1 federal district within the US were monitored for the occurrence of L. pneumophila Sg1, from January 2009 to December 2010 (Figure 1). At each site, one (n = 12) or two (n = 28) cold
Figure 1. Geographical map of United States - location and site type in study. △ Indicates site as a house and ■ indicates site as a building. The number insert indicates the number of taps at the site.
water taps were selected for monitoring. In total 68 taps - 29 kitchen sinks, 21 bathroom sinks, 17 drinking water fountains, and 1 refrigerator door water dispenser were monitored. The water analyzed was used as the home or building potable water source. No site had a secondary water treatment device installed, with the exception of the refrigerator door which had an in-line filter. The monitoring of two taps per site allowed U.S. EPA to evaluate the extent of the exposure to L. pneumophila Sg1 within a site. 3146
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25 μL qPCR reaction, 5 μL of standard template was analyzed per qPCR reaction equating to 100 mL of the original volume. In Silico Analysis of the Lp16S and LpLPS Primers. The National Center of Biotechnology Information’s (NCBI) primer-BLAST using Primer3 software http://www.ncbi.nlm. n ih .g o v/ t o o l s/ p ri m e r -b la s t / i n d e x . c g i? L I N K _ L O C = BlastHome28 and SeqMatch (version 3) from Ribosomal Database Project (RDP) http://rdp.cme.msu.edu/seqmatch/ seqmatch_intro.jsp29 were utilized (in silico) to determine specificity of the Lp16S and LpLPS primers and probe sets. Quantitative PCR (qPCR) Conditions. Two primer probes sets were used to detect and quantify L. pneumophila and L. pneumophila Sg1 in water. All DNA extracts were analyzed using the Lp16S primer-probes set, which is specific for L. pneumophila. If the extract was positive for this target, the second primer-probe set LpLPS, which is specific for L. pneumophila serogroup 1 (Sg1), was used to confirm the L. pneumophila detection as well as provide Sg-specific information. The qPCR reaction targeting the 16S rRNA gene contained the following: 17 μL of TaqMan Universal PCR Master Mix, 2.0 μL of a mixture of forward and reverse primers (500 nM) and a 100 nM IDT Probe, 1 μL of 0.1% Bovine Serum Albumin (BSA), and 5 μL of DNA purified. The Lp16S primer-probes set targeted a 100 basepair (bp) sequence of the 16s rRNA gene was developed using TaqMan chemistry, in-house.30 The probe and primers (IDT, Corelville, IA) for the Lp16S assay were the following: • probe Lpneu P1: 5′-6FAM-AAGCCCAGGAATTTCACAGAT −TAMRA • forward primer, Lpneu F1: 5′-CGGAATTACTGGGCGTAAAGG-3′ • reverse primer, Lpneu R1: 5′-GAGTCAACCAGTATTATCTGACCG-3′ Reactions were performed in triplicate 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 45 cycles of 10 s at 95 °C, 30 s at 60 °C, 1 s at 72 °C, and a cooling step of 30 s at 40 °C. A sample was considered positive for L. pneumophila if two or more of the replicates had a quantification cycle (Cq) value 5 × 103 Genomic Unit (GU)/L and >4 × 103 GU/L of L. pneumophila was recommend for cooling towers and hot and cold water systems, respectively.47 By applying this suggested alert level to the Lp16S data set in this study, only four taps exceed the proposed alert limits. Disinfectants Used in Potable Water Treatment. Disinfectants such as chlorine and monochloramine are used to reduce the indigenous microbes in source water. In recent years, studies have shown Legionella colonization within a water system is significantly reduced when a municipality converts to the use of monochloramine as its disinfectant.31,36 In a retrospective cohort study of 15 hospitals, there was a strong statistical association between disinfection with monochloramine and absence of Legionella in the water.48 Monochloraminated water was also found to produce a statistically significant reduction in the risk for hospital-acquired legionellosis.49 Of the 68 taps sampled in this study, 66 taps received treated water from a public utility. Based on the retrieved water quality reports, 43 taps (63%) received chlorinated water and 23 taps
Figure 3. A) Distribution of the concentration genomic target determined by each assay, LpLPS and Lp16S, for all taps. B) Distribution of the concentration genomic target determined by each assay, LpLPS and Lp16S, for all taps that show a colonization of L. pneumophila. The LQ bar shown in part A is for the LpLPS assay. *Analytical sensitivity of the assays was different. 3149
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monochloramine locations.50 This is most likely due to the broad geographic area and multiple sampling episodes covered by this study. In our study, two qPCR assays were evaluated and found suitable for the detection and quantification of L. pneumophila and L. pneumophila Sg1 in potable water. Over two years, 68 taps were each monitored four times. L. pneumophila Sg1 was detected in 47% of the taps. In the majority (31%) of cases, only one sample from a given site tested positive. There were a total of 11 taps that showed colonization, but only five reached concentrations that exceeded a > 4 × 103 GU/L qPCR action level for L. pneumophila Sg1as suggested by Lee et al. (2011).47 This study looked at L. pneumophila Sg1 occurrence on a limited but national scale. It provided evidence that exposure to L. pneumophila Sg1, the species/serogroup etiologic agent attributed to most legionellosis cases,51 occurs at point of use taps that provide potable water to workers and families. This study demonstrates that home and facility water distribution lines can become a vehicle for exposure to Legionella when aerosolized. Over the past decade, the incidence of legionellosis has increased in some areas across the US, especially the Northeast/Middle Atlantic regions.52 Collier et al. estimated that 433 million dollar per year is spent for the hospitalization of legionellosis cases.53 The present study demonstrated that gene products of L. pneumophila Sg1 are occurring and colonizing at point of use taps at locations across the U.S. Further research is needed to identify biotic and/or abiotic factors that contributed to L. pneumophila Sg 1 occurrence at the locations evaluated.
(37%) received water treated with monochloroamine. Frequency of detection of L. pneumophila Sg1 in a sample was similar for both disinfectant types. However, taps classified as colonized based on more than one detection were observed more frequently from systems that used chlorine (7/43, 16%) than systems that used chloramine (2/23, 8.6%). Statistical analysis of concentration (genomic target/L) data did not find a significant difference between the chlorinated and chloraminated samples. In general, monochloramine does not reduce the occurrence but may have an effect on the concentration of L. pneumophila Sg1 (Figure 4). The spread
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Figure 4. Distribution of L. pneumophila Sg1 concentrations (genomic target/L) by disinfectant. Data used came from the LpLPS assay alone (n = number of taps).
ASSOCIATED CONTENT
S Supporting Information *
Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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of median concentrations of L. pneumophila Sg1 per tap showed that concentrations in water treated with monochloramine had a lower variability. Also, the 75th percentile monochloramine value is a full log lower than that for the chlorine treatment. Whether these trends reflect real differences caused by disinfection treatment type will require studies with greater statistical power. However, the data suggest that monochloramine may be a better disinfectant for L. pneumophila Sg1 in potable water. In Figure 4, 75% of the samples from monochloraminated water were well below the LD for culture. This suggests that the significant drop in detection frequency identified in studies by Moore et al. (2006) and Flannery et al. (2006) was due to the concentration of L. pneumophila dropping below the LD for culture.36 31 Partial evidence to support this hypothesis is found in the report by Wang et al. (2012). They revisited some of the sites studied by Moore et al. (2006) and detected L. pneumophila in 5.6% of the water samples and at 20% of the sites using a qPCR technique compared to the 6.2% of sites reported by Moore et al. (2006). Unfortunately, Wang et al. (2012) only reported the highest concentration and the average concentration for water samples making it difficult to determine the percentage of samples with concentration below the culture LD.50 Overall, L. pneumophila Sg1 was molecularly detected in 19.5% (18/92) of the water samples from this study disinfected with monochloramine. This frequency of detection is larger than what Wang et al. (2012) reported for both their
AUTHOR INFORMATION
Corresponding Author
*Phone: 513-569-7634. Fax: 513-569-7757. E-mail: Donohue.
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We sincerely thank all those who participated in this study. The authors would also wish to acknowledge Dr. Helen Buse and Jill Hoelle for sharing their Legionella culture collection with us. The United States 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.
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dx.doi.org/10.1021/es4055115 | Environ. Sci. Technol. 2014, 48, 3145−3152
