Sulfate Reducing Bacteria and Mycobacteria Dominate the Biofilm

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Sulfate Reducing Bacteria and Mycobacteria Dominate the Biofilm Communities in a Chloraminated Drinking Water Distribution System Christa Kimloi Gomez-Smith, Timothy M. LaPara, and Raymond M Hozalski Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00555 • Publication Date (Web): 22 Jun 2015 Downloaded from http://pubs.acs.org on June 23, 2015

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Environmental Science & Technology

Sulfate Reducing Bacteria and Mycobacteria Dominate the Biofilm Communities in a Chloraminated Drinking Water Distribution System

C. Kimloi Gomez-Smith1,2, Timothy M. LaPara1, 3, Raymond M. Hozalski1,3*

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Department of Civil, Environmental, and Geo- Engineering, University of Minnesota, Minneapolis, Minnesota 55455 United States

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Water Resources Sciences Graduate Program, University of Minnesota, St. Paul, Minnesota 55108, United States 3

BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108, United States

Inquiries to: Raymond M. Hozalski, Department of Civil, Environmental, and Geo- Engineering, 500 Pillsbury Drive SE, Minneapolis, MN 554555, Tel: (612) 626-9650. Fax: (612) 626-7750. Email: [email protected]

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ABSTRACT The quantity and composition of bacterial biofilms growing on ten water mains from a

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full-scale chloraminated water distribution system were analyzed using real-time PCR targeting

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the 16S rRNA gene and next-generation, high-throughput Illumina sequencing. Water mains

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with corrosion tubercles supported the greatest amount of bacterial biomass (n = 25; geometric

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mean = 2.5 × 107 copies cm-2), which was significantly higher (P = 0.04) than cement-lined cast-

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iron mains (geometric mean = 2.0 × 106 copies cm-2). Despite spatial variation of community

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composition and bacterial abundance in water main biofilms, the communities on the interior

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main surfaces were surprisingly similar, containing a core group of operational taxonomic units

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(OTUs) assigned to only 17 different genera. Bacteria from the genus Mycobacterium dominated

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all communities at the main wall-bulk water interface (25% to 78% of the community),

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regardless of main age, estimated water age, main material, and the presence of corrosion

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products. Further sequencing of the mycobacterial heat shock protein (hsp65) provided species-

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level taxonomic resolution of mycobacteria. The two dominant Mycobacteria present, M.

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frederiksbergense (arithmetic mean = 85.7% of hsp65 sequences) and M. aurum (arithmetic

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mean = 6.5% of hsp65 sequences), are generally considered to be non-pathogenic. Two

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opportunistic pathogens, however, were detected at low numbers: M. haemophilum (arithmetic

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mean = 1.5% of hsp65 sequences) and M. abscessus (arithmetic mean = 0.006% of hsp65

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sequences). Sulfate-reducing bacteria from the genus Desulfovibrio, which have been implicated

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in microbially-influenced corrosion, dominated all communities located underneath corrosion

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tubercules (arithmetic mean = 67.5% of the community). This research provides novel insights

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into the quantity and composition of biofilms in full-scale drinking water distribution systems,

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which is critical for assessing the risks to public health and to the water supply infrastructure.

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INTRODUCTION

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Drinking water distribution systems (DWDSs) are an integral, although often overlooked,

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component in the provision of safe drinking water to consumers. Drinking water infrastructure is

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extensive, with more than 800,000 miles of main in the United States.1 Despite the multi-faceted

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approaches that water utilities employ to control microbial growth within the distribution system,

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including limiting availability of assimilable organic carbon2 and maintenance of a disinfectant

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residual, these systems are known to harbor substantial quantities of viable bacteria.3 Most of

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these bacteria (~95%) reside in biofilms on the walls of the water mains,4 which are comprised

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of a variety of materials, including unlined cast-iron, steel, ductile iron, cement-lined iron,

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various plastics, or wood.5 There is evidence that these biofilms may increase corrosion rates and

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affect main integrity,6-7 decrease residual disinfectant levels,8-10 and act as reservoirs for

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pathogens and other water quality-compromising bacteria. 11-12

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For the past few decades, research on DWDSs has focused on the study of pathogens and

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nitrifying bacteria in controlled experiments using laboratory-scale or simulated distribution

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systems.13-20 These studies used pipe loops, annular reactors, or other model systems to yield

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important information on the positive association of biomass levels with iron surfaces and the

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extent of corrosion,13, 15, 18 the effects of water chemistry (e.g., chlorine, sulfate, assimilable

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organic carbon) on biomass levels and community composition,18 and the ecology of the biofilm

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communities.17, 19 Clearly, there are limitations with using such laboratory-scale systems,

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perhaps the most significant of which is the relatively short operating time (i.e., months to a few

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years) in comparison to the service life of full-scale water mains (many decades). In contrast,

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Kelly et al.21 observed significant temporal variation in the biofilm community composition in

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water mains from a full-scale DWDS that correlated with water quality changes including

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fluctuations in residual chloramine concentration.

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In the present study, we determined the quantity (using quantitative PCR of 16S rRNA

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genes) and community composition (using Illumina MiSeq analysis on PCR-amplified 16S

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rRNA gene fragments) of bacteria growing on the internal surfaces of drinking water mains of

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varying type and age from a full-scale chloraminated DWDS. We hypothesized that corrosion

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tubercles found on cast-iron mains would harbor greater quantities of bacteria and specific

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bacterial populations known to contribute to corrosion as compared to cement-lined mains,

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which should harbor few (if any) corrosion-associated microbes. Substantial quantities of

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biomass were found on all types of water mains, but significantly higher biomass densities were

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detected beneath corrosion tubercles. The biofilm communities were of modest diversity

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(Shannon index: H′= 1.4 to 3.8) but exhibited surprisingly low evenness, as the communities

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living on the surface of the water mains were dominated by Mycobacterium-like organisms while

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the biofilms found underneath corrosion tubercles were dominated by Desulfovibrio-like

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organisms. Because many Mycobacteria spp. are pathogens or opportunistic pathogens, the gene

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encoding a 65-kilodalton mycobacterial heat shock protein (hsp65) was also characterized to

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more carefully resolve the Mycobacteria-like subcommunity.22-24

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MATERIALS AND METHODS

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Study Site. For this investigation, water main samples were collected from a single

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distribution system owned and operated by Saint Paul Regional Water Services (SPRWS) in

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Saint Paul, Minn. The main water source is the Mississippi River, which is first passed through a

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chain of lakes before entering the treatment plant. The water treatment process includes

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coagulation, lime softening, flocculation, sedimentation, recarbonation, filtration and

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disinfection. SPRWS treats an average of 132,000 m3 /day (35 × 106 gallons/day), although the

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maximum treatment capacity is 545,000 m3/day (144 × 106 gallons/day). There are

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approximately 1,770 kilometers (1,100 miles) of water main within the system and above-ground

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tanks that provide a water storage capacity of 496,000 m3 (131 × 106 gallons). The water is

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supplied to residents of the city and a few surrounding communities and consistently meets all

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applicable regulations. A summary of water quality monitoring results (i.e., total chlorine, pH,

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and total organic carbon) for the finished water is provided in the Supporting Information (Table

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S1).

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Water Quality Analyses. All water quality analyses were performed by SPWRS

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personnel as part of their routine sampling and monitoring. Total chlorine concentrations were

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determined by the N,N-diethyl-para-phenylenediamine (DPD) method25 using a Hach DR500

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UV-Vis spectrophotomer (finished water) or a LaMotte handheld colorimeter (distribution

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system). Monochloramine concentrations were determined using the Indophenol Method.26

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Sample Collection and DNA Extraction. Water main sections (length: 30-45 cm,

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diameter: 15 cm) were obtained from the distribution system in conjunction with routine

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replacement activities. The water main samples included: 8 cast iron water mains ranging in age

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from 64 to 129 years and 2 cement-lined cast iron water mains (53 and 57 years) (Table 1).

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Water ages for each water main were estimated using the InfoWater (Innovyze, Colorado)

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modeling program. Water main exteriors were cleaned using a chain cleaner prior to removal.

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Water main sections were manually extracted using a hinged cutter (Reed Manufacturing, Erie,

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PA, USA); the open ends were immediately sealed with sterile bags and then placed in a cooler

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and transported to the University of Minnesota. The water mains differed dramatically in

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appearance (Fig. 1); seven of the cast-iron water mains (primarily older) exhibited large tubercles

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while the other water mains (i.e., cement-lined and the newer, unlined cast-iron main) exhibited

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no obvious signs of tuberculation.

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Biofilms were sampled from the interiors of the water mains within 2 hours of water

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main extraction using a sterile metal spatula at least 10 cm from a cut end to minimize the risk of

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contamination. In cast-iron water mains with corrosion tubercles, the exterior surfaces of the

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tubercles were scraped (Tuberculated-Surface) and then tubercles were pried up and the loose

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solids underneath the tubercles were also collected (Under Tubercle). For the water mains

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without tuberculation (Non-Tuberculated cast-iron and Cement-Lined cast-iron), only surface

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samples were collected. Biofilm samples were collected from three locations in each water main,

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except Main 1, where 7 locations were sampled. In total, 51 samples of biofilm and associated

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loosely-bound corrosion products, weighing between 0.04 and 0.75 g (wet weight), were

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obtained (6 Cement Lined, 3 Non-Tuberculated, 27 Tuberculated-Surface, and 15 Under

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Tubercle). The plan area of each sampling site within the water mains was measured using

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digital calipers and ranged from 2 to 58 cm2 per sample. All samples were stored at -20°C prior

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to DNA extraction.

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DNA was extracted from the biofilm samples using the FastDNA Spin Kit for Soil (MP

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Biomedicals, Solon, OH, USA) per the manufacturer’s instructions. Briefly, ~0.5 g of sample

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(water main scraping and associated biofilm) was placed in a lysing matrix tube, immersed in

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CLS-TC buffer (MP Biomedicals), and rigorously shaken for 30 seconds in a FastPrep bead-

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beating instrument (FastPrep, Savant) on the 5.5 m/s mix setting. Extracted and purified DNA

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was stored at -20oC.

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PCR and Illumina MiSeq Analysis. PCR targeting the V3 region of the 16S rRNA gene was performed using primers 338F27 (5′-ID-ACT CCT ACG GGA GGC AGC AG-3′) and

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518R28 (5′-ID-ATT ACC GCG GCT GCT GG-3′) as described previously.29-30 Similarly, the

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hsp65 gene was amplified using primers tb11 (5′-ID-ACC AAC GAT GGT GTG TCC AT-3′)

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and tb12 (5′-ID-CTT GTC GAA CCG CAT ACC CT-3′) as described previously.31 For both

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sets of PCR primers, ID is an Illumina adapter sequence including a six- or eight-base

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multiplexing identification barcode unique to each sample.29 PCR products were initially

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screened using 2% agarose gels and then purified using the QIAquick Gel Extraction kit

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(Qiagen; Valencia, Calif.) per manufacturer’s instructions. Purified PCR products were pooled

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in equal concentrations and used as template for paired-end sequence analysis (2 × 150bp for

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16S rRNA genes; 2 × 300bp for hsp65 genes) on a MiSeq Desktop Sequencer (Illumina, Inc.;

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San Diego, Calif.) at the University of Minnesota Genomics Center (UMGC). Sequences are

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available through GenBank under BioProject PRJNA 273966.

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Quantitative PCR. Quantitative real-time polymerase chain reaction (qPCR) was

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performed using an Eppendorf Mastercycler ep realplex thermal cycler (Eppendorf, Westbury,

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NY, USA) to quantify 16S rRNA genes as a measure of total bacterial biomass as described

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previously.32 Briefly, each qPCR run consisted of initial denaturation for 10 min at 95°C,

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followed by 40 cycles of denaturation at 95°C for 15 s, and anneal/extension at a target-specific

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temperature for 1 min. A 25 µL reaction mixture contained 12.5 µL of iTaq SYBR Green

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Supermix with ROX (Bio-Rad; Hercules, Calif.), 25 µg bovine serum albumin (Roche Applied

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Science; Indianapolis, Ind.), optimized quantities of forward and reverse primers (to limit the

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formation of primer-dimers), and 0.5 µL of template DNA. The quantity of target DNA in

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samples was calculated based on comparison to standard curves generated using known

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quantities of plasmid DNA containing the 16S rRNA gene of E. coli K12. Standard curves

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consisted of at least five different dilutions of plasmid DNA run during each qPCR (r2 > 0.99). 6 ACS Paragon Plus Environment

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The amplification curves of individual samples were compared to those of the standards to

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ensure that the samples and standards amplified with similar efficiencies (amplification

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efficiencies were 100 ± 8%). Negative controls obtained during sampling were also quantified in

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order to account for background contamination. All negative controls had Cq values > 26 cycles,

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whereas all water main samples had Cq values < 22 cycles.

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Data Analysis. The statistical analyses were performed on log-transformed 16S rRNA

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gene quantities using JMP Pro ver. 11.2.0 (SAS Institute; Cary, N.C.). One-way analysis of

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variance (ANOVA) was initially performed to determine if statistically significant differences

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existed in the data set; Tukey’s honesty significant difference (HSD) test was then used to

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determine the statistical significance of differences between the means of pairwise comparisons.

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Illumina MiSeq sequence data was processed and analyzed using Mothur v.1.29.2.33 For

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all sequences, paired-end reads were initially combined to give a single sequence, after which the

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sequences were screened for quality. Sequences containing any mismatches with the barcoded

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primers, sequence lengths outside of the expected range for the target gene (135-151 bp for 16S

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rRNA gene fragments; 400-500 bp for hsp65 fragments), ambiguous bases, or homopolymers

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longer than 8 bp were excluded. In addition, sequences were removed if the MiSeq-defined

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average quality score was less than 35 (Q35 = 99.97% accuracy in base calling) over a sliding

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window of 50 bp. Qualifying sequences were binned by sample; primers and barcodes were

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trimmed from the sequencing reads.

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For 16S rRNA genes, sequences were aligned to the SILVA bacterial 16S rRNA

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database; chimeric sequences were removed using the UCHIME algorithm34 and the results were

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randomly subsampled to 44,455 sequences (i.e., the number of sequences in the sample with the

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least number of sequences) to avoid bias due to variable coverage of each sample. Sequences

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were clustered into operational taxonomic units (OTUs) at a cutoff of 99% sequence identity.

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Taxonomic information for all sequences was obtained using the RDP database release 9.35

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Weighted unifrac distances36 were computed for non-metric multidimensional scaling (nMDS)

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analysis.

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For hsp65 sequences, sequences were aligned to a newly-created database containing

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hsp65 sequences from 200 strains of Mycobacteria spp. and several other phylogenetically-

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related organisms (see Supporting Information, Table S2). Sampled sequences were aligned to

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the alignment reference file and preclustered into groups of 95% sequence identity. Sequences

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were then filtered, matched to the taxonomic reference file, and clustered into OTUs at a cutoff

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of 95% sequence identity. Bootstrap values > 60% were sufficient for species-level

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classification.

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RESULTS

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Ten water mains of different ages were collected from various locations throughout the

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DWDS during April 16-September 23, 2013 (see Supporting Information, Figure S1). Eight of

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the water mains were comprised of unlined cast-iron, with 7 of the 8 exhibiting tuberculation.

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The remaining two water mains were cement-lined cast-iron and exhibited no tuberculation.

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Distribution System Water Quality. Finished water leaving the SPRWS treatment plant

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contained a total chlorine residual (arithmetic mean ± standard deviation) of 3.5 ± 0.2 mg/L, and

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a mean TOC concentration of 4.2 ± 0.8 mg/L during the water main sampling period (April 16 -

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September 23, 2013). The mean total chlorine residual for DWDS water samples (n = 1,113)

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obtained from 85 locations throughout the distribution system was 2.6 ± 0.4 mg/L. For these

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DWDS samples, the mean pH was 9.0 ± 0.3, with 99.8% of samples within the pH range of 8.5

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to 9.4. Additional water quality data of the finished water leaving the SPRWS treatment plant

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effluent and from water collected from the DWDS during the water main sampling period are

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summarized in the Supporting Information (Table S1).

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Quantification of Bacterial Biomass. Substantial quantities of bacterial biomass,

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measured as 16S rRNA gene copies, were detected on the surfaces of all water mains as well as

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underneath the corrosion tubercules (Fig. 2). Of the surface samples, the Tuberculated Surface

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had the highest quantity of biomass (n = 25; geometric mean = 2.5 × 107 copies cm-2; geometric

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standard deviation = 1.0), which was significantly greater (P = 0.04) than the quantity of biomass

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on the Cement-Lined mains (n = 6; geometric mean = 2.0 × 106 copies cm-2; geometric standard

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deviation = 0.6) but not statistically greater (P = 0.13) than the quantity of biomass on the Non-

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Tuberculated main (n = 3; geometric mean = 1.6 × 106 copies cm-2; geometric standard deviation

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= 0.7). The highest levels of bacterial biomass were detected in the Under Tubercle samples (n

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= 15; geometric mean = 5.0 × 109 copies cm-2; geometric standard deviation = 0.7), and the mean

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was significantly higher (P < 0.0001) than that for any other sample type. Finally, biomass

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values did not correlate with main age or the estimated water age at a given main location.

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Bacterial Community Composition. The bacterial community composition of the

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biofilms growing on the surfaces of all of the water mains as well as underneath the corrosion

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tubercles was determined by Illumina MiSeq profiles of PCR-amplified 16S rRNA gene

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fragments. A total of 18,291,384 sequences were obtained, which were screened for quality and

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then sub-sampled to 42,445 sequences per profile so that community analysis could be

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performed without bias related to the depth of sequence information for each sample. All

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sequences analyzed were classified as Bacteria, with a total of 33,474 different OTUs among all

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of the samples. All OTUs were further classified to the genus level with 100% bootstrap

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confidence, with no unassigned OTUs. Although OTUs were assigned to 1,217 different genera,

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OTUs attributed to only 18 genera comprised the majority (86.8%) of all sequences.

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Multiple samples were initially collected from seven different locations within Main 1

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(Tuberculated Surface) (Fig. 3A) and from three different locations within Main 2 (Tuberculated

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Surface) (Fig. 3B) to help ascertain the relative reproducibility of the Illumina MiSeq profiling

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pipeline. In both cases, there were substantial differences in Illumina MiSeq profiles within

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Main 1 and within Main 2, which could have been caused by genuine differences in bacterial

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community composition or due to random variation in the Illumina MiSeq profiling pipeline. To

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help resolve this question, one of the samples from Main 2 was processed in triplicate, giving

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highly reproducible results (Fig. 3B), suggesting that the observed variation in all of the samples

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is due to genuine variation in bacterial community composition rather than random variation

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implicit to the Illumina MiSeq pipeline.

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The water main biofilm communities exhibited relatively low diversity as quantified by

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the Shannon index (Table 2). More specifically, the biofilm communities exhibited a reasonably

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high degree of richness (as suggested by the ACE richness estimate) but most of the

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communities were very uneven, with a single bacterial population comprising a very large

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fraction (> 50%) of the community. The most abundant OTUs within all of the water main

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surface communities were of the genus Mycobacterium (Fig. 4A). The fraction of

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Mycobacterium-like OTUs varied from as low as 24.8% ± 1.7% (Main 5) to as high as 77.7% ±

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4.0% (Main 10); these OTUs were detected at similar levels whether the water main was

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Tuberculated (51.7% ± 21.2%), Non-Tuberculated (60.2% ± 9.8%), or Cement-Lined (73.1% ±

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6.1%). In contrast, Mycobacterium-like OTUs comprised only a small portion of the Under

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Tubercle bacterial communities (1.8% ± 2.7%). The most abundant OTUs in the Under

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Tubercle communities were of the genus Desulfovibrio (67.5% ± 26.6%) (Fig 4B). These

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Desulfovibrio-like OTUs were also detected in the water main surface samples, but at much

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lower abundances (Tuberculated: 13.7% ± 18.5%; Cement-Lined: 0.2% ± 0.1%; Non-

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Tuberculated: 0.3% ± 0.3%).

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Identification of Mycobacterial Strains. Because the Mycobacteria contain several

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known and opportunistic pathogens,37 PCR and Illumina MiSeq analysis was used to profile a

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461bp fragment of a gene encoding a 65-kilodalton heat shock protein, the sequence of which

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has been reported to give species-level resolution of Mycobacteria.31 Although PCR

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amplification of hsp65 was attempted on all 51 water main samples, only 19 amplified

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adequately for sequence analysis. The number of sequences obtained for Under Tubercle (n = 5),

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Cement-Lined (n = 2), and Non-tuberculated (n = 1) samples ranged from 1,365 to 9,336 per

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sample, while Tuberculated Surface (n = 11) samples had the largest and most variable number

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of sequences, ranging from 7,712 to 29,972 per sample. A total of 15,779 OTUs were classified

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as 72 different mycobacterial species (see Supporting Information for a complete list, Table S3).

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Ninety-six percent of Mycobacterium-like OTUs had a 100% bootstrap confidence; 2% of OTUs

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had a 61-99% bootstrap confidence; and 2% of OTUs had a bootstrap confidence below 60% and

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were thus labeled “unclassified.” Although the lack of sufficient replicates from each of the

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samples thwarted statistical analysis, the specific populations of Mycobacteria appeared

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substantially different among the different water main types (Table 3). The most common

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hsp65 sequence in all water main types was associated with M. frederiksbergense, ranging from

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~80% of the community (Non-Tuberculated and Under Tubercle) to as high as ~99% of the

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community (Cement-Lined). M. aurum-like sequences were the second most abundant OTU in

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the Tuberculated Surface samples, whereas M. haemophilum was the second-most abundant

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OTU in the Non-Tuberculated sample (11.3%). The Under Tubercle samples, however, had

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several different Mycobacteria spp. (M. lentiflavum, M. psychrotolerans, M. holsaticum, M.

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nebraskense, and M. haemophilum) comprise anywhere from 1% to 5% of the hsp65 profiles.

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DISCUSSION

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This manuscript provides novel information on the quantities and community

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composition of bacteria growing on water mains from a full-scale distribution system.

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Information of this type is exceptionally rare because of the cost and inconvenience of exhuming

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and analyzing such water mains. Hence, most of the prior research in this area investigated

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bench- or pilot-scale simulated water distribution systems, used surrogates for water mains (e.g.,

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coupons, water meters), or sampled tap water. A limitation of the research described herein is

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that it focused solely on a single distribution system with a stable chloramine residual throughout

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its extensive network. Additional research is needed to characterize the bacterial communities

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growing in a wide variety of distributions systems, such that the roles of disinfectant type,

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residual disinfectant concentration, and other factors can be better understood.

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The abundance of Desulfovibrio-like OTUs underneath all sampled corrosion tubercles

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suggests that microbial-influenced corrosion is a significant contributor to the deterioration of

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unlined cast-iron drinking water mains. Desulfovibrio spp. are sulfate-reducing bacteria (SRB)

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that have been shown to cause localized corrosion and shorten the design life of various

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materials in built environments.38 Research on SRB on the interior of water mains dates back to

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at least the 1940s,39-40 where their presence was believed to exacerbate corrosion. These early

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investigations and more recent studies41-44 clearly demonstrate the abundance of SRBs in

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corroded iron water mains. Despite aerobic conditions in the bulk water, the DWDS biofilms and

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corrosion tubercles provide anaerobic niches where sulfate-reducing bacteria can proliferate.

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Additionally, there is ample evidence that some Desulfovibrio spp., although classified as

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obligate anaerobes, are equipped with thioredoxins that enable them to survive exposure to oxic

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environments.45-46

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Our results show that Desulfovibrio-like OTUs dominated the bacterial community

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underneath corrosion tubercles in quantities that greatly exceed previous cultivation-dependent

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quantifications of sulfate-reducing bacteria.41-42 Their location underneath corrosion tubercles not

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only affords these organisms protection from dissolved oxygen and chlorine in the bulk water but

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also can provide the SRB with access to molecular hydrogen as electron donor from the

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reduction of protons during the iron corrosion process. The consumption of hydrogen is one of

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several proposed microbial-influenced corrosion mechanisms attributed to SRB.7, 47 The

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prevalence of Desulfovibrio-like OTUs in the DWDS environment and their implication in

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corrosion in other environments suggests that attempts to limit their growth, such as eliminating

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the supply of sulfate, may be beneficial. Nevertheless, more work is needed to elucidate the role

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of Desulfovibrio spp. in iron water main degradation, as the mere association of Desulfovibrio

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spp. with corrosion tubercles is not conclusive evidence of their role in the corrosion process.7, 48

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There have been numerous reports of the occurrence of Mycobacteria in water

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distribution system biofilms.49-56 Our study extends these findings by showing that

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Mycobacteria-like OTUs were prominent in all biofilms growing on the surface of water mains

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within a chloraminated DWDS, regardless of main age, main type, and main location.

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Mycobacteria spp. have a variety of characteristics that afford them a competitive advantage in a

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chloraminated water distribution system, including increased resistance to disinfectants in

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comparison to many other bacteria,57 the ability to form biofilms, and their ability to survive in

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nutrient-poor conditions.58-59 Furthermore, there is recent experimental evidence from a

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simulated distribution system study to suggest that monochloramine, rather than free chlorine,

341

selects for Mycobacteria over other genera.60 From this perspective, the abundance of

342

Mycobacteria may actually be a useful indicator of a stable chloramine residual, but more

343

research is needed to determine the prevalence of these organisms in full-scale distribution

344

systems with a different disinfectant (e.g., free chlorine) or without a residual disinfectant.

345

In contrast, the presence of Mycobacteria spp. within DWDS could be considered

346

undesirable due to the ability of some Mycobacteria spp., specifically the Mycobacterium avium

347

complex (MAC), to cause infection in immunocompromised persons. By profiling the gene

348

encoding a 65-kilodalton heat-shock protein (hsp65), we determined that no MAC-like OTUs

349

were present in the DWDS. The two dominant Mycobacterium-like OTUs from all sampling

350

dates and mains were M. frederiksbergense (83.3%) and M. aurum (11.4%). Although M.

351

frederiksbergense has been isolated in association with a catheter-related infection61 and two

352

subcutaneous injection-related infections,62 this Mycobacterium sp. is not generally recognized

353

as a pathogen. Similarly, M. aurum is generally considered to be nonpathogenic. Although M.

354

aurum was previously identified in association with two cases of infection,63-64 the accuracy of

355

these identifications have been questioned following the discovery that American Type Culture

356

Collection M. aurum strains were misannotated.65 Rather, the infection-associated bacterium

357

probably belonged to the opportunistic M. neoaurum-“M. lacticola” group.65 A few known

358

opportunistic Mycobacteria spp. (M. haemophilum, M. abscessus, M. xenopi) were detected in

359

the DWDS, however, they were rare members of the mycobacterial communities (0.013-11%, 0-

360

0.11%, 0-0.15%, respectively), with M. abscessus identified in only one sample, and M. xenopi

361

detected in 6 samples. Thus, the risk of direct exposure to harmful Mycobacteria via tap water

362

from shedding of water main biofilms appears to be minimal. Nevertheless, additional research is

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363

needed to clarify this issue because there is the potential for harmful Mycobacteria spp. to

364

multiply in hot water systems as the optimum growth temperature for many Mycobacteria is in

365

the range of 28 to 38.5°C.66

366

Despite differences in main type, age, and location within the distribution system, water

367

main biofilm communities on interior surfaces were surprisingly similar, with OTUs assigned to

368

only 17 different genera comprising the majority of sequences for these samples. Observed inter-

369

and intra-main variation in composition was due primarily to differences in the relative

370

abundance of community members rather than to differences in the specific community members

371

present, which has been previously observed.67 The similarity of all of the surface biofilm

372

communities throughout the DWDS is consistent with the relatively stable water quality

373

conditions in the system.

374

Highly corroded water mains were capable of supporting higher densities of bacteria

375

overall, in comparison to non-tuberculated and cement-lined mains. Corrosion tubercles

376

potentially provide niches where bacteria can be sheltered from the bulk water flow as well as

377

increased surface area for biofilm growth.68 The presence of corrosion deposits may additionally

378

shield bacteria from disinfectant residuals by exerting a disinfectant demand.3, 14-15 Furthermore,

379

nutrients have been shown to concentrate in iron tubercles69-70 and in corrosion scales,71 where

380

they are available for bacterial growth. Consequently, reducing the presence of corrosion

381

deposits within the water mains, either by decreasing corrosion rates or using corrosion resistant

382

materials (e.g., cement-lined iron or plastic) may be effective at limiting bacterial growth.15

383

Although corroded water mains generally supported more biomass, there were large variations in

384

bacterial densities in a given main, especially for the heavily tuberculated mains. Patchy biofilm

385

coverage in drinking water distribution systems is consistent with previous studies17, 56, 72-74 and

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386

suggests that the suitability of biofilm growth conditions in water mains varies substantially over

387

small distances.

388

In conclusion, this research provides novel insights into the composition of biofilms in a

389

full-scale chloraminated drinking water distribution system, which is critical for assessing the

390

risks to public health and critical water supply infrastructure. The predominance of

391

Mycobacteria-like OTUs on water main surfaces and Desulfovibrio-like OTUs underneath

392

tubercles has implications for the management and assessment of water distribution systems.

393

Although it is impractical to completely eradicate bacteria from distribution systems, it may be

394

beneficial to instead focus on methods to manipulate the bacterial community composition for a

395

desirable result. For example, the association of sulfate-reducing Desulfovibrio spp. with

396

corrosion tubercles suggests that corrosion prevention efforts should include approaches for

397

limiting growth of sulfate-reducing bacteria, such as decreasing sulfate concentrations in the

398

finished water. In addition, non-pathogenic Mycobacteria spp. might be a viable candidate for

399

the “pro-biotic” distribution system strategy described by Wang et al.75 Finally, quantification of

400

Mycobacteria spp. in DWDS biofilms potentially could be used to assess the long-term stability

401

of the chloramine residual.

402

ACKNOWLEDGEMENTS

403

Financial support for this work was provided by the Board of Water Commissioners of

404

the City of Saint Paul. We thank David Schuler, Jim Bode, Kou Vang, Richard Hibbard, Renee

405

Huset, and CheFei Chen for helping coordinate this study and for providing access to the water

406

mains, water quality data, and other necessary information.

407

SUPPORTING INFORMATION

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408

The Supporting Information gives additional information on the water quality from the

409

DWDS during this study (Table S1), the reference database used to analyze hsp65 (Table S2),

410

the complete list of hsp65 sequences detected in this study (Table S3), a map of the sample

411

locations used in this study (Figure S1), and a non-metric multidimensional analysis plot of

412

weighted unifrac distances between communities (Figure S2). This information is available free

413

of charge via the Internet at http://pubs.acs.org/.

414

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415 416 417 418

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61. Senozan, E. A.; Adams, D. J.; Giomanco, N. M.; Warwick, A. B.; Eberly, M. D. A catheterrelated blood stream infection with Mycobacterium frederiksbergense in an immunocompromised child. Pediatr. Infect. Dis. J. 2015, 34(4), 445-447.

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74. Van der Kooij, D.; Veenendaal, H. R.; Baars-Lorist, C.; Van der Klift, D. W.; Drost, Y. C. Biofilm formation on surfaces of glass and teflon exposed to treated water. Water Res. 1995, 7, 1655-1662.

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75. Wang, H., Edwards, M. A.; Falkinham, J. O.; Pruden, A. Probiotic approach to pathogen control in premise plumbing systems? A review. Environ. Sci. Technol. 2013, 47 (18), 10117-10128.

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Table 1. Description of the water main samples collected in this study and the associated estimated water ages at each sampling location. Main

Sampling Date

Main Age (years)

Main Material

Water Age (hrs)

1 2 3 4 5 6 7 8 9 10

4/17/13 5/16/13 6/27/13 8/19/13 8/19/13 9/4/13 9/23/13 6/27/13 6/20/13 7/25/13

127 90 127 129 129 64 86 48 51 53

Unlined Cast-iron Unlined Cast-iron Unlined Cast-iron Unlined Cast-iron Unlined Cast-iron Unlined Cast-iron Unlined Cast-iron Unlined Cast-iron Cement-lined cast-iron Cement-lined cast-iron

19 20 21 60 60 70 72 16 22 88

633 634

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635 636 637 638 639 640 641

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Table 2. The number of observed OTUs, the number of predicted OTUs (ACE estimator), and Shannon index for bacterial communities obtained from full-scale water mains via Illumina MiSeq analysis of PCR-amplified 16S rRNA gene fragments. Sequence libraries consisted of 42,445 sequences per profile after trimming randomly subsampling to normalize the number of sequences. All values represent the arithmetic mean (± one standard deviation) of replicate communities obtained from each main (n = 7 for Main 1; n = 3 for all other Mains). Tuberculated Surface

Non-tuberculated Cement-Lined Under Tubercle

Main 1 2 3 4 5 6 7 8 9 10 3 4 5 6 7

Observed OTUs 1888 ± 405 1915 ± 186 1989 ± 148 1374 ± 52 1774 ± 98 1512 ± 45 1773 ± 208 2692 ± 296 2233 ± 110 1906 ± 52 1706 ± 51 1492 ± 78 1294 ± 380 1588 ± 14 967 ± 65

ACE Estimator 11767 ± 3102 12300 ± 347 12315 ± 1830 8005 ± 765 8205 ± 1452 11197 ± 2543 7556 ± 2332 6518 ± 1920 9406 ± 1983 10567 ± 285 9532 ± 584 7894 ± 1142 6673 ± 3778 8523 ± 1448 8229 ± 1720

642 643

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Shannon Index 2.4 ± 0.5 2.2 ± 0.3 1.9 ± 0.5 2.0 ± 0.3 3.5 ± 0.0 2.9 ± 0.3 2.8 ± 0.5 3.3 ± 0.7 2.3 ± 0.1 1.7 ± 0.2 2.0 ± 0.6 1.8 ± 0.5 2.3 ± 0.5 2.8 ± 0.1 1.6 ± 0.3

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Table 3. The percent abundances (arithmetic mean ± standard deviation) of Mycobacterium species represented in each water main type. Only abundant (>1% of a single sample) species are reported here. Illumina Miseq sequencing of the hsp65 gene was used to give varying amounts of sequences per sample (ranging from 1,365 to 29,972 sequences per sample). Percent Abundance by Sample Type (Mean ± standard deviation) Mycobacterium sp.

M. frederiksbergense M. aurum M. haemophilum M. lentiflavum M. psychrotolerans M. holsaticum M. nebraskense

Tuberculated Surface (n=11) 86.0 ± 15.9 10.6 ± 15.2 1.0 ± 1.9 0.2 ± 0.8 0.009 ±0.02 0.007 ± 0.01 0.01 ± 0.02

Cement-Lined (n=2) 98.9 ± 1.1 0.1 ± 0.1 0.7 ± 0.9 0±0 0±0 0±0 0.02 ± 0.03

NonTuberculated (n=1) 78.9 1.7 11.3 0 0 0.5 0

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Under Tubercle (n=5) 80.9 ± 13.7 0.9 ± 0.7 1.1 ± 1.2 4.2 ± 3.3 3.9 ± 5.1 2.7 ± 5.3 1.2 ± 1.8

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Figure Captions

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Fig. 1. Photographs of representative water mains analyzed in this study. (A) Tuberculated

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unlined cast-iron, (B) Non-tuberculated unlined cast-iron, and (C) Cement-lined cast-iron.

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Fig. 2. Total biomass of main community samples as indicated by quantitative real-time PCR of

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the 16S rRNA gene per cm2 of scraped biofilm. Abbreviations refer to sample type and main

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type: TS-Tuberculated surface, UT-Under tubercle, NT- Non-tuberculated, CL-Cement lined.

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Fig. 3. Bacterial community profiles from two tuberculated cast-iron water mains as determined

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by Illumina MiSeq analysis of PCR-amplified 16S rRNA gene fragments. Profiles reveal

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differences in bacterial community composition within a single water main. Main 2 replicates

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(A1, A2, A3) were obtained from the same sample/DNA extract, but were amplified by PCR and

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sequenced separately.

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Fig. 4. Bacterial community profiles of the 18 most abundant genera represented in (A) main

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surface samples, and (B) under tubercles samples as determined by Illumina MiSeq analysis of

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PCR-amplified 16S rRNA gene fragments. Abundant genera are those that comprised greater

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than 0.5% of total sequences for all samples combined (2,164,695 total sequences). Genera that

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represented less than 0.5% of total sequences are grouped in “Other.” Distribution profiles

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represent the arithmetic mean of triplicate communities obtained from each main, except for

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Main 1 (n = 7). Abbreviations refer to the type of water main: TS = Tuberculated surface, UT =

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Under tubercle, NT = Non-tuberculated, CL = Cement lined.

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