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Legionella DNA Markers in Tap Water Coincident with Spike in Legionnaires’ Disease in Flint, MI David Schwake, Emily D. Garner, Owen R. Strom, Amy Pruden, and Marc A. Edwards Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.6b00192 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016
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Legionella DNA Markers in Tap Water Coincident with Spike in Legionnaires’ Disease in
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Flint, MI
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David Otto Schwake1, Emily D. Garner1, Owen R. Strom1, Amy Pruden1, and Marc A. Edwards1*
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AUTHOR ADDRESS. 1Via Department of Civil and Environmental Engineering, Virginia Tech,
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418 Durham Hall, Blacksburg, VA 24061, United States
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*CORRESPONDING AUTHOR. Email:
[email protected]; Phone: (540) 231-7236; Fax: (540) 231-7916
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ABSTRACT
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Two clusters of Legionnaires’ Disease occurred in Flint, MI subsequent to switching to a
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corrosive potable water source from April 2014-October 2015. We hypothesized that the
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interrupted corrosion control and associated release of iron, nutrients, and depleted chlorine
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residual in the distribution system would lead to high levels of Legionella. Tap water surveyed
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throughout Flint in August and October 2015 confirmed L. pneumophila in two hospitals (mean
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1,890 ± 2,220 gene copy numbers/mL, 48% positivity), but not small single-story buildings. The
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hospitals frequently had optimal Legionella growth temperatures and were located in high water
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age zones of the distribution system (3 d to >6 d). Relatively high concentrations of iron were
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present (mean 51.0 ± 37.2 ppb) and Cl2 residual was sporadic (mean 0.700 ± 0.775 mg/L)
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throughout the Flint distribution system. This study addresses knowledge gaps linking
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legionellosis outbreaks to changes in municipal water quality and distribution system operation.
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INTRODUCTION
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In April 2014, the City of Flint, located in Genesee County Michigan, began to use the
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local Flint River as their drinking water source (chlorinated, with no corrosion inhibitor), instead
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of the municipal water that they had purchased for decades from the City of Detroit (Lake Huron
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water, chlorinated and treated with orthophosphate for corrosion inhibition).1 Failure to
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implement a federally mandated corrosion-control program triggered an array of water quality
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issues, including rampant corrosion and lead contamination throughout the distribution system.
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Other consequences included increased frequency of main breaks,2 rapid loss of disinfectant
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residual,2 and high levels of total trihalomethanes.3 Microbial aspects of water quality also
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deteriorated, with elevated fecal coliform bacteria and multiple boil-water advisories.2 After a
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public health emergency was declared, Flint returned to Detroit water in October, 2015.
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In January 2016, it was announced that cases of Legionnaires’ Disease, a deadly
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pneumonia caused by Legionella bacteria (usually L. pneumophila), had spiked in Genesee
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County during the period of Flint River usage.4,5 45 cases with 5 deaths occurred from June
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2014 through March 2015,4 and 46 cases with 7 deaths from May through October 2015.5 A
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high percentage of cases occurred in people receiving Flint drinking water at home or that had
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visited a Flint hospital (65/91),4,5 suggesting the outbreaks may have been tied to the municipal
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water system.
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The water switch and subsequent legionellosis cases in Flint, MI presented an
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opportunity to investigate linkages between municipal water quality and abundance of
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Legionella in premise plumbing. Here we report the findings of two field surveys of single story
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homes/businesses and multi-storied hospitals in Flint, conducted during August and October
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2015, while the drinking water was still sourced from the Flint River. Levels of DNA markers
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corresponding to Legionella spp. and L. pneumophila (23S rRNA and mip gene copies/mL,
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respectively) are compared to similar US surveys carried out in absence of documented
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outbreaks6,7 and considered alongside various physiochemical characteristics of the water to
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provide insight into contributing factors in the Flint outbreaks and inform strategies for
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minimizing the likelihood of waterborne disease.
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MATERIALS AND METHODS
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Study Site and Water Sampling
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Two surveys of tap water in Flint, MI and vicinity were conducted in 2015. The first,
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occurring August 18-19, focused on small buildings: 16 single-story homes and businesses
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within Flint and 4 businesses within nearby Flint Township (which was maintained on Detroit
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water). The second, occurring October 15-16, focused on two health care centers in Flint
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(Hospitals 1 and 2), immediately prior to the city switching back to Detroit water. These
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hospitals were selected due to their location in high-water age zones in the distribution system
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and because large institutional buildings are known to be particularly susceptible to Legionella
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colonization.8
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Cold water samples were collected from all buildings in the first survey, with hot water
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additionally sampled in three Flint homes. Hot and cold samples were collected in the second
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survey, from public restroom hand wash sinks, representing a cross section of various buildings
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and floors from both hospitals. A total of 60 samples were collected from 30 outlets in three
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buildings in Hospital 1, while 40 samples were collected from 20 outlets in Hospital 2.
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First flush tap samples of approximately 1,000 mL were collected for microbiological
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analysis in sterile polypropylene bottles pre-treated with 24 mg sodium thiosulfate and 292 mg
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ethylenediaminetetraacetic acid (in solution, adjusted to pH 8.5) and kept on ice until processing.
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Subsequently, two 10 mL cold water samples were collected in polyethylene tubes for
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measurements of chlorine, analyzed immediately, and metals, analyzed later following storage at
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room temperature. All microbiological samples were either shipped overnight or transported by
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ground the same day to Virginia Tech. These procedures were modified slightly for hospital
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sampling to include additional sample collection. Two samples were collected at each tap,
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starting with a first flush cold water sample. After this, the hot water lines were flushed for 30
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seconds, followed by collection of hot water.
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Water Quality Analysis
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Outlet water temperature and flow rate were measured at the time of sample collection.
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Total chlorine (detection limit 0.1 mg/L) was measured in field using a DR2700 portable
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spectrophotometer (Hach, Loveland, CO). Metals, including iron (10 ppb detection limit), were
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measured by inductively coupled plasma mass spectrometry according to Standard Method
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3125B.9 Samples were not filtered prior to analysis, yielding a measurement of total metals
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(dissolved+particulate).
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Sample Processing and Quantitative Polymerase Chain Reaction (q-PCR)
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Within ~24 hours of sample collection, 1,000 mL water samples were concentrated onto
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sterile 0.22 µm pore size mixed cellulose esters membranes (Millipore, Billerica, MA). Sample
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bottles were weighed before and after filtration to determine the volume filtered. Filters were
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torn to pieces, transferred to DNA extraction tubes, and stored at -20 °C until extraction using
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FastDNA SPIN Kits (MP Biomedicals, Solon, OH). q-PCR reaction was used to quantify
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Legionella spp. 23S rRNA and L. pneumophila mip genes following previously established
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methods10,11 on a CFX96 Real-Time System (Bio-Rad, Hercules, CA). Further details on q-PCR
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methods are available in the SI.
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Data Analysis
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Two-tailed Mann-Whitney Tests tested for significant differences in data distributions
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(p) (accessed June 29, 2016). 27. Michigan Department of Environmental Quality. City of Flint Water Treatment Plant Monthly Operating Report August 2013 (http://www.michigan.gov/flintwater/0,6092,7-345-377816--,00.html) (accessed June 29, 2016). 28. Cianciotto, N.P. Iron acquisition by Legionella pneumophila. Biometals. 2007, 20, 323-331. 29. Bargellini, A.; Marchesi, I.; Righi, E.; Ferrari, A.; Cencetti, S.; Borella, P.; Rovesti, S. Parameters predictive of Legionella contamination in hot water systems: association with trace elements and heterotrophic plate counts. Water Res. 2011, 45, (6), 2315-2321. 30. Kraemer, S.M. Iron oxide dissolution and solubility in the presence of siderophores. Aquatic Sciences. 2004, 66 (1), 3-18.
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31. Beer, K.D.; Gargano, J.W.; Roberts, V.A.; Hill, V.R.; Garrison, L.E.; Kutty, P.K.; Hilborn, E.D.; Wade, T.J.; Fullerton, K.E.; Yoder, J.S. Surveillance for waterborne disease outbreaks associated with drinking water – United States, 2011-2012. CDC Morbidity and Mortality Weekly Report. 2015, 64, 842-848. 32. Dooling, K.L; Toews, K.; Hicks, L.A.; Garrison, L.E.; Bachaus, B.; Zansky, S.; Carpenter, R.; Schaffner, B.; Parker, E.; Petit, S.; et. al. Active bacterial core surveillance for legionellosis – United States, 2011-2013. CDC Morbidity and Mortality Weekly Report. 2015, 64 (42), 11901193. 33. Collier, S.A.; Stockman, L.J.; Hicks, L.A.; Garrison, L.E.; Zhou, F.J.; Beach, M.J. Direct healthcare costs of selected diseases primarily or partially transmitted by water. Epidemiol. Infect. 2012, 140 (11), 2003-2013. 34. Wang, H.; Masters, S.; Falkinham, J.O.; Edwards, M.A.; Pruden, A. Distribution system water quality affects responses of opportunistic pathogen gene markers in household water heaters. Environ. Sci. Technol. 2015, 49 (14), 8416-8424. 35. Pruden, A.; Edwards, M.A.; Falkinham, J.O. State of the science and resarch needs for opportunistic pathogens in premise plumbing. Water Research Foundation. 2013. 36. Davis, M.M.; Kolb, C.; Reynolds, L.; Rothstein, E.; Sikkema, K. Flint Water Advisory Task Force-Final Report. 2016 (https://www.michigan.gov/documents/snyder/FWATF_FINAL_REPORT_21March2016_5178 05_7.pdf) (accessed June 29, 2016). 37. Ditommaso, S.; Ricciardi, E.; Giacomuzzi, M.; Arauco Rivera, S.R.; Zotti, C.M. Legionella in water samples: how can you interpret the results obtained by quantitative PCR. Mol. Cell Probes. 2015, 19 (10), 7-12. 38. Legionella pneumophila: Dose Response Models. (http://qmrawiki.canr.msu.edu/index.php/Legionella_pneumophila:_Dose_Response_Models) (accessed May 6, 2016). 39. Lee, J.V.; Lai, S.; Exner, M.; Lenz, J.; Gaia, V.; Casati, S.; Hartemann, P.; Lück, C.; Pagnon, B.; Ricci, M.L.; et. al. An international trial of quantitative PCR for monitoring Legionella in artificial water systems. J. Appl. Microbiol. 2011, 110 (4), 1032-1044. 40. Joly, P.; Falconnet, P.; André, J.; Weill, N.; Reyrolle, M.; Vandenesch, F.; Maurin, M.; Etiene, J.; Jarraud, S. Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl. Environ. Microbiol. 2006, 72 (4) 2801-2808. 41. US Environmental Protection Agency. Safety of Public Water Systems (Safe Drinking Water Act). Updated 2002.
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TABLES AND FIGURES
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Table 1: Occurrence of Legionella spp. and Legionella pneumophila gene copies in various
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Flint, MI locationsa and comparison to published surveys of tap water
Legionella Concentration Range (GC/mL) Legionella Mean Concentration (GC/mL ± SD) L. pneumophila Concentration Range (GC/mL)e L. pneumophila Mean Concentration (GC/mL ± SD)e Legionella Positivity (%) L. pneumophila Positivity (%) Mean Chlorine Concentration (mg/L ± SD) Mean Temperature (°C ± SD) Municipal Water Age Range (Hrs) Mean Iron Concentration (ppb)
Hospital 2 Hot Water (n=20)
Hospital 2 Cold Water (n=19)
Flint Small Buildings (n=19)
Flint Township Small Buildings (n=4)
Virginiad (n=90)
US Cold Water Surveyc (n=269)
Floridad (n=54)
Hospital 1 Hot Water (n=29)
Hospital 1 Cold Water (n=30)
42.8 – 66000
32.2 – 119000
119 – 5170
28.5 – 61300
11.9 – 2460
ND
NR
10.4-2300
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73-144
73-144
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NR
NR
72 - >408
72 - >408
NR
37.6 ± 32.3
NR
52.9 ± 25.3
72.3 ± 46.4
0 ±0
NR
NR
NR
f
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a
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represent only quantifiable data; NR- Not reported, no data available, or not assayed, ND:
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Assayed, but not detected; cDonohue et al. 20147; dWang et al. 20126, used identical q-PCR
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methodology in the same laboratory as the current study; eL. pneumophila carries one copy of the
Mean Legionella and L. pneumophila values for Flint hospital and small building samples
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mip gene,31 thus, gene copy numbers and GU are interchangeable; fValues represent data for L.
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pneumophila Serogroup 1 in GU/mL.
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Figure 1. Concentrations of Legionella spp. and L. pneumophila gene copies as determined by q-
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PCR in water samples collected from public tap water outlets in two Flint, MI hospitals (A:
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Hospital 1, B: Hospital 2). One hot and one cold water sample were collected from each outlet.
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Outlets with no data points correspond to samples with concentrations below the limit of
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quantification.
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